Combination therapy to treat Mycobacterium diseases

ABSTRACT

The present invention relates to a compound of formula (I) 
                         
or a pharmaceutically acceptable salt thereof wherein X and R are as defined herein. The compounds of formula (I) are useful as gyrase and/or topoisomerase IV inhibitors for treating bacterial infections. The compounds of formula (I) either possess a broad range of anti-bacterial activity and advantageous toxicological properties or are prodrugs of compounds having said activity.

RELATED APPLICATIONS

This application is a non-provisional filing which claims the benefit ofProvisional U.S. Patent Application Ser. No. 61/673,109, filed Jul. 18,2012 and Provisional U.S. Patent Application Ser. No. 61/782,496, filedMar. 14, 2013. The entire contents of these patent applications arehereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Mycobacterium tuberculosis, the bacterium which causes tuberculosis(TB), remains a major cause of death in the world in spite of relativelyeffective treatments with multi-drug combinations due to the lack oftreatment accessibility, HIV co-infection, and the lengthy treatmenttime. Combinations of multiple drugs are required to prevent theemergence of drug resistance and to effectively treat the infection.Drug sensitive TB is treated with isoniazid, rifampicin, pyrazinamideand ethambutol or streptomycin. Importantly, there are many side effectsassociated with the drugs making compliance difficult and tolerabilitychallenging. For example, isoniazid causes peripheral nervous systemdisorder and induces serious liver dysfunction in some people when usedin combination with rifampicin; rifampicin can cause liver dysfunctionor hepatopathy, malaise, drug allergy, and its use with other drugs suchas HIV protease inhibitors, is compromised due to P450-associated enzymeinduction. In summary, tolerability remains a major challenge forcurrent TB drug treatment regimens.

A revitalized effort to develop new drug treatments and combinations ofdrugs has been inspired by medical philanthropy and cooperative effortsof public private partnerships. In addition, in spite of the totalnumber of cases of TB having decreased in most countries, the lack ofaccessibility of treatment of multi-drug resistant (MDR-TB) andextensively drug resistant (XDR-TB) combined with the emergence ofextremely drug resistant (XXDR-TB) or totally drug resistant (TDR-TB)raises global concerns about the sustainability of the current controlmeasures. Furthermore, there remain too few combinations that have thepotential to shorten the treatment time of drug sensitive and drugresistant TB infection (currently from six months to two years,respectively), which remains critical to treatment compliance andpreventing the emergence of drug resistance. Moreover, there ismechanistic and chemical redundancy for compounds currently in clinicaldevelopment and safety liabilities for most classes of drugs reflectinga lack of bold and innovative approaches. For example, twofluoroquinolones in phase 3 clinical trials do not address the problemof XDR-TB and may exacerbate drug resistance; three oxazolidinones inphase 2 clinical trials have not proven to shorten the time oftreatment; and two nitroimidazoles in clinical development may beincompatible with some of the front line treatment drugs. Thus, thereremains an overall dearth of drug candidates for the treatment oftuberculosis; see WGND pipeline as referencehttp://www.newtbdrugs.org/pipeline.php.

As a result of the need to combat drug-resistant bacteria and theincreasing failure of the available drugs, there has been a resurgentinterest in discovering new antibiotics. One attractive strategy fordeveloping new antibiotics is to inhibit DNA gyrase and/or topoisomeraseIV, bacterial enzymes necessary for DNA replication, and therefore,necessary for bacterial cell growth and division. Gyrase and/ortopoisomerase IV activity are also associated with events in DNAtranscription, repair and recombination. Gyrase is one of thetopoisomerases, a group of enzymes which catalyze the interconversion oftopological isomers of DNA (see generally, Kornberg and Baker, DNAReplication, 2d Ed., Chapter 12, 1992, W. H. Freeman and Co.; Drlica,Molecular Microbiology, 1992, 6, 425; Drlica and Zhao, Microbiology andMolecular Biology Reviews, 1997, 61, pp. 377-392). Gyrase itselfcontrols DNA supercoiling and relieves topological stress that occurswhen the DNA strands of a parental duplex are untwisted during thereplication process. Gyrase also catalyzes the conversion of relaxed,closed circular duplex DNA to a negatively superhelical form which ismore favorable for recombination. The mechanism of the supercoilingreaction involves the wrapping of gyrase around a region of the DNA,double strand breaking in that region, passing a second region of theDNA through the break, and rejoining the broken strands. Such a cleavagemechanism is characteristic of a type II topoisomerase. The supercoilingreaction is driven by the binding of ATP to gyrase. The ATP is thenhydrolyzed during the reaction. This ATP binding and subsequenthydrolysis cause conformational changes in the DNA-bound gyrase that arenecessary for its activity. It has also been found that the level of DNAsupercoiling (or relaxation) is dependent on the ATP/ADP ratio. In theabsence of ATP, gyrase is only capable of relaxing supercoiled DNA.

Bacterial DNA gyrase is a 400 kilodalton protein tetramer consisting oftwo A (GyrA) and two B subunits (GyrB). Binding and cleavage of the DNAis associated with GyrA, whereas ATP is bound and hydrolyzed by the GyrBprotein. GyrB consists of an amino-terminal domain which has the ATPaseactivity, and a carboxy-terminal domain which interacts with GyrA andDNA. By contrast, eukaryotic type II topoisomerases are homodimers thatcan relax negative and positive supercoils, but cannot introducenegative supercoils. Ideally, an antibiotic based on the inhibition ofbacterial DNA gyrase and/or topoisomerase IV would be selective forthese enzymes and be relatively inactive against the eukaryotic type IItopoisomerases.

Topoisomerase IV primarily resolves linked chromosome dimers at theconclusion of DNA replication.

The widely-used quinolone antibiotics inhibit bacterial DNA gyrase(GyrA) and/or Topoisomerase IV (ParC). Examples of the quinolonesinclude the early compounds such as nalidixic acid and oxolinic acid, aswell as the later, more potent fluoroquinolones such as norfloxacin,ciprofloxacin, and trovafloxacin. These compounds bind to GyrA and/orParC and stabilize the cleaved complex, thus inhibiting overall gyrasefunction, leading to cell death. The fluoroquinolones inhibit thecatalytic subunits of gyrase (GyrA) and/or Topoisomerase IV (Par C) (seeDrlica and Zhao, Microbiology and Molecular Biology Reviews, 1997, 61,377-392). However, drug resistance has also been recognized as a problemfor this class of compounds (WHO Report, “Use of Quinolones in FoodAnimals and Potential Impact on Human Health”, 1998). With thequinolones, as with other classes of antibiotics, bacteria exposed toearlier compounds often quickly develop cross-resistance to more potentcompounds in the same class. The associated subunits responsible forsupplying the energy necessary for catalytic turnover/resetting of theenzymes via ATP hydrolysis are GyrB (gyrase) and ParE (topoisomeraseIV), respectively (see, Champoux, J. J., Annu. Rev. Biochem., 2001, 70,pp. 369-413). Compounds that target these same ATP binding sites in theGyrB and ParE subunits would be useful for treating various bacterialinfections (see, Charifson et al., J. Med. Chem., 2008, 51, pp.5243-5263).

There are fewer known inhibitors that bind to GyrB. Examples include thecoumarins, novobiocin and coumermycin A1, cyclothialidine, cinodine, andclerocidin. The coumarins have been shown to bind to GyrB very tightly.For example, novobiocin makes a network of hydrogen bonds with theprotein and several hydrophobic contacts. While novobiocin and ATP doappear to bind within the ATP binding site, there is minimal overlap inthe bound orientation of the two compounds. The overlapping portions arethe sugar unit of novobiocin and the ATP adenine (Maxwell, Trends inMicrobiology, 1997, 5, 102).

For coumarin-resistant bacteria, the most prevalent point mutation is ata surface arginine residue that binds to the carbonyl of the coumarinring (Arg136 in E. coli GyrB). While enzymes with this mutation showlower supercoiling and ATPase activity, they are also less sensitive toinhibition by coumarin drugs (Maxwell, Mol. Microbiol. 1993, 9, 681).

Despite being potent inhibitors of gyrase supercoiling, the coumarinshave not been widely used as antibiotics. They are generally notsuitable due to their low permeability in bacteria, eukaryotic toxicity,and poor water solubility (Maxwell, Trends in Microbiology, 1997, 5,102). It would be desirable to have a new, effective GyrB and ParEinhibitor that overcomes these drawbacks and, preferably does not relyon binding to Arg136 for activity. Such an inhibitor would be anattractive antibiotic candidate, without a history of resistanceproblems that plague other classes of antibiotics.

As bacterial resistance to antibiotics has become an important publichealth problem, there is a continuing need to develop newer and morepotent antibiotics. More particularly, there is a need for antibioticsthat represent a new class of compounds not previously used to treatbacterial infection. Compounds that target the ATP binding sites in boththe GyrB (gyrase) and ParE (topoisomerase IV) subunits would be usefulfor treating various bacterial infections. Such compounds would beparticularly useful in treating nosocomial infections in hospitals wherethe formation and transmission of resistant bacteria are becomingincreasingly prevalent. Furthermore, there is a need for new antibioticshaving a broad spectrum of activity with advantageous toxicologicalproperties.

SUMMARY OF THE INVENTION

The present invention is directed to a method of controlling, treatingor reducing the advancement, severity or effects of a mycobacteriumdisease comprising administering to a patient in need thereof atherapeutically effective amount of gyrase and/or topoisomerase IVinhibitors of formula

wherein R is hydrogen or fluorine;

-   X is hydrogen, —PO(OH)₂, —PO(OH)O⁻M⁺, —PO(O⁻)₂.2M⁺, or —PO(O⁻)₂.D²⁺;    M⁺ is a pharmaceutically acceptable monovalent cation; and D²⁺ is a    pharmaceutically acceptable divalent cation; or a pharmaceutically    acceptable salt thereof; in combination with one or more antibiotic    compounds comprising a diarylquinolone, rifapentine, rifalazil, a    nitroimidazole, a benzothiazinone, capreomycin, clofazimine,    cycloserine, dapsone, a thiocarbamide, ethambutol, DC-159a, a    nitrobenzthiazole, sutezolid (PNU-100480), AZD-5847, posizolid    (AZD-2563), para-aminosalicylic acid, SQ-109, SQ-609, a capuramycin,    a caprazene nucleoside, an isothiazoloquinolone, thioridazine,    thiacetazone, dirithromycin, roxithromycin, telithromycin,    azithromycin, clarithromycin, erythromycin, amikacin, kanamycin,    streptomycin, levofloxacin, moxifloxacin, gatifloxacin, linezolid,    rifalazil, meropenem, clavulanate, or isoniazid, with the proviso    that when the one or more antibiotic compounds is thioridazine,    azithromycin, clarithromycin, erythromycin, amikacin, kanamycin,    streptomycin, levofloxacin, moxifloxacin, gatifloxacin, linezolid,    rifalazil, meropenem, clavulanate, and isoniazid, then an additional    antibiotic is also present in the combination.

In one embodiment, the one or more antibiotic compounds comprisesbedaquiline (TMC-207), delaminid (OPC67683), PA 824, TBA-354, SKLB-TB37,BTZ-043, SQ-641, cycloserine, dapsone, ethionamide, prothionamide,para-aminosalicylic acid, CPZEN45, ACH-702 or ACH-710. In anotherembodiment, the one or more antibiotic compounds comprises bedaquiline,rifapentine, moxifloxacin, linezolid, delaminid, or PA 824. In afurtherr embodiment, the one or more antibiotic compounds comprisesmoxifloxacin, linezolid, rifalazil, meropenem, clavulanate,pyrazinamide, and isoniazid. In some embodiments, the combinationfurther comprises pyrazinamide.

In one embodiment, the mycobacterial disease is caused by M.tuberculosis, M. avium intracellulare, M. ulcerans, M. kansasii, M.fortuitum, M. abcesses, M. leprae, M. africanum, M. marinum, M. aviumparatuberculosis, or M. bovis, M. chelone, M. scrofulaceum, M. xenopi,M. intracellulare, or M. micron. In a further embodiment, themycobacterium disease is tuberculosis. In some embodiments, the compoundof formula (I) is administered with only one antibiotic selected fromrifapentine, TMC-207, SQ-109, a nitroimidazole, or an oxazolidinone.

In one embodiment, the present invention is directed to a method ofinhibiting the growth of drug sensitive and drug resistant mycobacteriacells wherein the mycobacteria cell is drug sensitive and drug resistantM. tuberculosis, drug resistant M. tuberculosis, M. aviumintracellulare, M. ulcerans, M. kansasii, M. fortuitum, M. abcesses, M.leprae, M. africanum, M. marinum, M. avium paratuberculosis, or M.bovis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a thermal ellipsoid plot of two symmetry independent moleculesof compound 12.

FIG. 2 is a thermal ellipsoid plot of two symmetry independent moleculesof compound 23.

DETAILED DESCRIPTION

The present invention is directed to gyrase and/or topoisomerase IVinhibitors and pharmaceutically acceptable salts thereof, in combinationwith one or more antibiotics and optionally pyrazinamide.

The present invention provides methods and compositions comprising thecompound of formula (I), or a pharmaceutically acceptable salt thereof,in combination with one or more antibiotic agents and, optionally,pyrazinamide effective in treatment and prevention of disease caused bymicroorganisms including, but not limited to, mycobacteria. Inparticular, the methods and compositions of the present invention areeffective in inhibiting the growth of the microorganism, M.tuberculosis.

As used herein, the term “tuberculosis” comprises disease states usuallyassociated with infections caused by mycobacteria species comprising M.tuberculosis complex. The term “tuberculosis” is also associated withmycobacterial infections caused by mycobacteria other than M.tuberculosis (MOTT). Other mycobacterial species include M.avium-intracellulare, M. kansasii, M. fortuitum, M. chelonae, M. leprae,M. africanum, and M. micron, M. avium paratuberculosis, M.intracellulare, M. scrofulaceum, M. xenopi, M. marinum, M. ulcerans.

The present invention further provides methods and compositions usefulfor the treatment of infectious disease, including but not limited to,tuberculosis, leprosy, Crohn's Disease, acquired immunodeficiencysyndrome, Lyme disease, cat-scratch disease, Rocky Mountain SpottedFever and influenza.

In one embodiment, the one or more antibiotic is selected from the groupconsisting with one or more antibiotic compounds comprising adiarylquinolone, rifapentine, rifalazil, a nitroimidazole, abenzothiazinone, capreomycin, clofazimine, cycloserine, dapsone, athiocarbamide, ethambutol, DC-159a, a nitrobenzthiazole, sutezolid(PNU-100480), AZD-5847, posizolid (AZD-2563), para-aminosalicylic acid,SQ-109, SQ-609, a capuramycin, a caprazene nucleoside, anisothiazoloquinolone, thioridazine, thiacetazone, dirithromycin,roxithromycin, telithromycin, azithromycin, clarithromycin,erythromycin, amikacin, kanamycin, streptomycin, levofloxacin,moxifloxacin, gatifloxacin, linezolid, rifalazil, meropenem,clavulanate, or isoniazid, with the proviso that when the one or moreantibiotic compounds is thioridazine, azithromycin, clarithromycin,erythromycin, amikacin, kanamycin, streptomycin, levofloxacin,moxifloxacin, gatifloxacin, linezolid, rifalazil, meropenem,clavulanate, and isoniazid, then an additional antibiotic is alsopresent in the combination.

The combinations of the present invention are effective against diseasecaused by infectious organisms, including mycobacteria. One embodimentof the invention provides methods and compositions comprising thecompound of formula (I), or a pharmaceutically acceptable salt thereof,in combination with one or more antibiotic agents and, optionally,pyrazinamide.

Another embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with one or more antibiotic agents andoptionally pyrazinamide that have MIC values of 50 μM or lower formycobacterial organisms. Another embodiment of the present inventioncomprises compositions that have an MIC value of 25 μM or lower formycobacterial organisms. Yet another embodiment of the present inventioncomprises compositions that have an MIC value of 12.5 μM or lower formycobacterial organisms. Another embodiment of the present inventioncomprises compositions that have an MIC value of 5 μM or lower formycobacterial organisms. In another embodiment of the present invention,the methods and compositions comprise the compound of formula (I), or apharmaceutically acceptable salt thereof, in combination with one ormore antibiotic agents and, optionally, pyrazinamide having FITS Lucactivity of 10% or greater.

The present invention contemplates treatment for animals, including, butnot limited to, humans. Thus, it is an object of the present inventionto provide methods and compositions for the treatment and prevention ofdiseases caused by mycobacteria such as tuberculosis.

Yet another object of the present invention is to provide methods andcompositions for the treatment and prevention of infectious diseasesusing compositions comprising the compound of formula (I), or apharmaceutically acceptable salt thereof, in combination with one ormore antibiotic agents and, optionally, pyrazinamide.

Another object of the present invention is to provide methods andcompositions for the treatment and prevention of mycobacterial diseaseusing compositions comprising the compound of formula (I), or apharmaceutically acceptable salt thereof, in combination with one ormore antibiotic agents and, optionally, pyrazinamide.

Still another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising the compound of formula (I), or apharmaceutically acceptable salt thereof, in combination with one ormore antibiotic agents and, optionally, pyrazinamide.

Another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising the compound of formula (I), or apharmaceutically acceptable salt thereof, in combination with one ormore antibiotic agents and, optionally, pyrazinamide, wherein thecomposition has an MIC value of 50 μM, or less.

Another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising the compound of formula (I), or apharmaceutically acceptable salt thereof, in combination with one ormore antibiotic agents and, optionally, pyrazinamide, wherein thecomposition has an MIC value of 25 μM, or less.

Another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising the compound of formula (I), or apharmaceutically acceptable salt thereof, in combination with one ormore antibiotic agents and, optionally, pyrazinamide, wherein thecomposition has an MIC value of 12.5 μM, or less.

Yet another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising the compound of formula (I), or apharmaceutically acceptable salt thereof, in combination with one ormore antibiotic agents and, optionally, pyrazinamide, wherein thecomposition has an MIC value of 5 μM, or less.

Yet another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising the compound of formula (I), or apharmaceutically acceptable salt thereof, in combination with one ormore antibiotic agents and, optionally, pyrazinamide, wherein thecomposition has HTS/Luc activity of 10% or greater.

Yet another object of the present invention is to provide compositionsfor therapeutic formulations for the treatment and prevention ofmycobacterial disease.

Another object of the present invention is to provide compositions fortherapeutic formulations for the treatment and prevention ofmycobacterial disease caused by mycobacterial species comprising M.tuberculosis complex, M. avium intracellulare, M. kansasii, M.fortuitum, M. chelonoe, M. leprae, M. africanum, M. micron, M. bovis BCGor M. bovis.

Still another object of the present invention is to provide compositionsand methods for the treatment or prevention of infectious disease causedby Mycobacterium-fortuitum, Mycobacterium marinum, Helicobacter pylori,Streptococcus pneumoniae and Candida albicans.

As used herein, the terms “in combination with” and “combinations” referto the use of two or more agents in one treatment regardless of whetherthe agents are in a single formulation or in multiple formulations. Theuse of the term “in combination with” and “combinations” do not restrictthe order in which treatments are administered to a subject beingtreated for a disease caused by a mycobacterium. The administration ofthe multiple agents may be simultaneous or sequential. A first treatmentcan be administered prior to, concurrently with, after, or within anycycling regimen involving the administration of a second or thirdtreatment to a subject with a disease or a condition caused by amycobacterium. For example, one treatment may be administered 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before one ormore treatments; or one treatment may be administered 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after one or moretreatments. Such treatments include, for example, the administration ofcompounds having Formula I in combination with pyrazinamide and one ormore antibiotic agents.

As used herein, the term “halogen” means F, Cl, Br, or I.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Isotopically-labeled forms of compounds of formula (I) wherein one ormore atoms are replaced by an atom having an atomic mass or mass numberdifferent from the atomic mass or mass number usually found in natureare also included herein. Examples of isotopes that can be incorporatedinto compounds of the invention includes isotopes of hydrogen, carbon,nitrogen, oxygen, and fluorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, and¹⁷O. Such radio-labeled and stable-isotopically labeled compounds areuseful, for example, as research or diagnostic tools or gyrase and/ortopoisomerase IV inhibitors with improved therapeutic profile. Thestructures also encompass zwitterionic forms of the compounds or salts,where appropriate.

The gyrase and/or topoisomerase IV inhibitors of the present inventionmay be represented by formula (I) or salts thereof:

wherein R is hydrogen or fluorine; X is hydrogen, —PO(OH)₂, —PO(OH)O⁻M⁺,—PO(O⁻)₂.2M⁺, or —PO(O⁻)₂.D²⁺; M⁺ is a pharmaceutically acceptablemonovalent cation; and D²⁺ is a pharmaceutically acceptable divalentcation. The compounds of formula (I) either possess abroad range ofanti-bacterial activity and advantageous toxicological properties or areprodrugs of compounds having said activity.

The gyrase and/or topoisomerase IV inhibitors of the present inventionmay be represented by formula (IA):

wherein R is hydrogen or fluorine. The compounds of formula (IA) possessa broad range of anti-bacterial activity and advantageous toxicologicalproperties.

The gyrase and/or topoisomerase IV inhibitors of the present inventionmay be represented by formula (IB):

wherein X is —PO(OH)₂, —PO(OH)O⁻M⁺, —PO(O⁻)₂.2M⁺, or —PO(O⁻)₂.D²⁺; M⁺ isa pharmaceutically acceptable monovalent cation; and D²⁺ is apharmaceutically acceptable divalent cation. The compounds of formula(IB) are phosphate ester prodrugs of the compound(R)-1-ethyl-3-(6-fluoro-5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea,which possesses a broad range of anti-bacterial activity andadvantageous toxicological properties.

In one embodiment, compounds of formula (I) include compounds of formula(IC)

wherein R is as defined above.

In another embodiment, compounds of formula (I) include compounds offormulae (ID) and (IE) as set forth below:

(R)-1-ethyl-3-(5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea,or a pharmaceutically acceptable salt thereof; and

(R)-1-ethyl-3-(6-fluoro-5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea,or a pharmaceutically acceptable salt thereof. Unless otherwise stated,the phrase “compounds of formula (I)” is intended to include otherformulae set forth herein that are encompassed by formula (I) includingformulae (IA), (IB), (IC), (ID), and (IE).

The compounds of formula (IB) are prodrugs of their parent compound,1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea.Thus, the activity exhibited upon administration of the prodrug isprincipally due to the presence of the parent compound that results fromcleavage of the prodrug.

An additional object of the present invention provides embodiments inwhich the one or more antibiotic compounds comprising a diarylquinolone,rifapentine, rifalazil, a nitroimidazole, a benzothiazinone,capreomycin, clofazimine, cycloserine, dapsone, a thiocarbamide,ethambutol, DC-159a, a nitrobenzthiazole, sutezolid (PNU-100480),AZD-5847, posizolid (AZD-2563), para-aminosalicylic acid, SQ-109,SQ-609, a capuramycin, a caprazene nucleoside, an isothiazoloquinolone,thioridazine, thiacetazone, dirithromycin, roxithromycin, telithromycin,azithromycin, clarithromycin, erythromycin, amikacin, kanamycin,streptomycin, levofloxacin, moxifloxacin, gatifloxacin, linezolid,rifalazil, meropenem, clavulanate, or isoniazid, with the proviso thatwhen the one or more antibiotic compounds is thioridazine, azithromycin,clarithromycin, erythromycin, amikacin, kanamycin, streptomycin,levofloxacin, moxifloxacin, gatifloxacin, linezolid, rifalazil,meropenem, clavulanate, and isoniazid, then an additional antibiotic isalso present in the combination. In one embodiment, the additionalantibiotic is pyrazinamide.

In one aspect, the present invention provides embodiments in which theone or more antibiotic compounds comprise a diarylquinolone,rifapentine, rifalazil, or a nitrobenzthiazole. In another aspect,pyrazinamide may also be present in the combination. In a furtherembodiment, the compound of formula (I) includes compounds encompassedby formula (I) including formulae (IA), (IB), (IC), (ID), and (IE). Inone aspect, the present invention provides embodiments in which the oneor more antibiotic compounds comprise a nitroimidazole, abenzothiazinone, capreomycin, clofazimine, cycloserine, dapsone, athiocarbamide, ethambutol, DC-159a, or a nitrobenzthiazole. In anotheraspect, pyrazinamide may also be present in the combination. In afurther embodiment, the compound of formula (I) includes compoundsencompassed by formula (I) including formulae (IA), (IB), (IC), (ID),and (IE).

In one aspect, the present invention provides embodiments in which theone or more antibiotic compounds comprise a nitroimidazole, abenzothiazinone, capreomycin, clofazimine, cycloserine, dapsone, athiocarbamide, ethambutol, or DC-159a. In another aspect, pyrazinamidemay also be present in the combination. In a further embodiment, thecompound of formula (I) includes compounds encompassed by formula (I)including formulae (IA), (IB), (IC), (ID), and (IE).

In one aspect, the present invention provides embodiments in which theone or more antibiotic compounds comprise capreomycin, clofazimine,cycloserine, dapsone, a thiocarbamide, ethambutol, DC-159a, or anitrobenzthiazole. In another aspect, pyrazinamide may also be presentin the combination. In a further embodiment, the compound of formula (I)includes compounds encompassed by formula (I) including formulae (IA),(IB), (IC), (ID), and (IE).

In one aspect, the present invention provides embodiments in which theone or more antibiotic compounds comprise a diarylquinolone,rifapentine, rifalazil, dapsone, a thiocarbamide, ethambutol, DC-159a,or a nitrobenzthiazole. In another aspect, pyrazinamide may also bepresent in the combination. In a further embodiment, the compound offormula (I) includes compounds encompassed by formula (I) includingformulae (IA), (IB), (IC), (ID), and (IE).

In another aspect, the present invention provides embodiments in whichthe one or more antibiotic compounds comprise sutezolid (PNU-100480),AZD-5847, posizolid (AZD-2563), para-aminosalicylic acid, SQ-109,SQ-609, a capuramycin, a caprazene nucleoside, an isothiazoloquinolone,thioridazine, thiacetazone, dirithromycin, roxithromycin, telithromycin,azithromycin, clarithromycin, erythromycin, amikacin, kanamycin,streptomycin, levofloxacin, moxifloxacin, gatifloxacin, linezolid,rifalazil, meropenem, clavulanate, or isoniazid, with the proviso thatwhen the one or more antibiotic compounds is thioridazine, azithromycin,clarithromycin, erythromycin, amikacin, kanamycin, streptomycin,levofloxacin, moxifloxacin, gatifloxacin, linezolid, rifalazil,meropenem, clavulanate, and isoniazid, then an additional antibiotic isalso present in the combination. In another aspect, the additionalantibiotic is pyrazinamide. In a further embodiment, the compound offormula (I) includes compounds encompassed by formula (I) includingformulae (IA), (IB), (IC), (ID), and (IE).

In another aspect, the present invention provides embodiments in whichthe one or more antibiotic compounds comprise sutezolid (PNU-100480),AZD-5847, posizolid (AZD-2563), para-aminosalicylic acid, SQ-109,SQ-609, a capuramycin, a caprazene nucleoside, an isothiazoloquinolone,thiacetazone, dirithromycin, roxithromycin, or telithromycin. In anotheraspect, pyrazinamide may also be present in the combination. In afurther embodiment, the compound of formula (I) includes compoundsencompassed by formula (I) including formulae (IA), (IB), (IC), (ID),and (IE).

In another aspect, the present invention provides embodiments in whichthe one or more antibiotic compounds comprise thioridazine,azithromycin, clarithromycin, erythromycin, amikacin, kanamycin,streptomycin, levofloxacin, moxifloxacin, gatifloxacin, linezolid,rifalazil, meropenem, clavulanate, or isoniazid. In another aspect,pyrazinamide may also be present in the combination. In a furtherembodiment, the compound of formula (I) includes compounds encompassedby formula (I) including formulae (IA), (IB), (IC), (ID), and (IE).

In one embodiment, the one or more antibiotics comprises bedaquiline(TMC-207), delaminid (OPC67683), PA 824, TBA-354, BTZ-043, SQ-641,cycloserine, dapsone, ethionamide, prothionamide, para-aminosalicylicacid, CPZEN45, ACH-702 or ACH-710.

An additional object of the present invention provides embodiments inwhich the one or more antibiotic agents is pyrazinamide in combinationwith moxifloxacin, linezolid, rifalazil, meropenem, clavulanate, orisoniazid.

In another embodiment, the oxazolidinone may be linezolid, Sutezolid(PNU-100480), 2-oxazolidone, torezolid, posizolid, eperezolid,radezolid, AZD-5847, or those described in U.S. Pat. Nos. 5,981,528 and6,605,630.

In another embodiment, the diarylquinoline is bedaquiline (TMC-207).

In a further embodiment, the nitroimidazole may be PA 824, delaminid(OPC-67683), or TBA-354.

In a further embodiment, the one or more antibiotics suitable for thecombinations of the present application comprise bedaquiline (TMC-207),delaminid (OPC67683), PA 824, TBA-354, BTZ-043, SQ-641, cycloserine,dapsone, ethionamide, prothionamide, para-aminosalicylic acid, CPZEN45,ACH-702 or ACH-710.

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with TMC-207. In a further embodiment, thecompound of formula (I) includes compounds encompassed by formula (I)including formulae (IA), (IB), (IC), (ID), and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with Rifapentine. In a further embodiment,the compound of formula (I) includes compounds encompassed by formula(I) including formulae (IA), (IB), (IC), (ID), and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with oxazolidinone. In a furtherembodiment, the compound of formula (I) includes compounds encompassedby formula (I) including formulae (IA), (IB), (IC), (ID), and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with a nitroimidazole. In a furtherembodiment, the nitroimidazole is delaminid. In a further embodiment,the compound of formula (I) includes compounds encompassed by formula(I) including formulae (IA), (IB), (IC), (ID), and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with SQ-109. In a further embodiment, thecompound of formula (I) includes compounds encompassed by formula (I)including formulae (IA), (IB), (IC), (ID), and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with TMC-207 and pyrazinamide. In a furtherembodiment, the compound of formula (I) includes compounds encompassedby formula (I) including formulae (IA), (IB), (IC), (ID), and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with rifapentine and pyrazinamide. In afurther embodiment, the compound of formula (I) includes compoundsencompassed by formula (I) including formulae (IA), (IB), (IC), (ID),and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with oxazolidinone and pyrazinamide. In afurther embodiment, the compound of formula (I) includes compoundsencompassed by formula (I) including formulae (IA), (IB), (IC), (ID),and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with nitroimidazole and pyrazinamide. In afurther embidmnet, the nitroimidazole is delaminid. In a furtherembodiment, the compound of formula (I) includes compounds encompassedby formula (I) including formulae (IA), (IB), (IC), (ID), and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with pyrazinamide and SQ-109. In a furtherembodiment, the compound of formula (I) includes compounds encompassedby formula (I) including formulae (IA), (IB), (IC), (ID), and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with TMC-207, SQ-109, and pyrazinamide. Ina further embodiment, the compound of formula (I) includes compoundsencompassed by formula (I) including formulae (IA), (IB), (IC), (ID),and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with clavulanate and meropenem. In afurther embodiment, the compound of formula (I) includes compoundsencompassed by formula (I) including formulae (IA), (IB), (IC), (ID),and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with rifapentine, SQ-109, and pyrazinamide.In a further embodiment, the compound of formula (I) includes compoundsencompassed by formula (I) including formulae (IA), (IB), (IC), (ID),and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with oxazolidinone, SQ-109, andpyrazinamide. In a further embodiment, the compound of formula (I)includes compounds encompassed by formula (I) including formulae (IA),(IB), (IC), (ID), and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with nitroimidazole, SQ-109, andpyrazinamide. In a further embodiment, the nitroimidazole is delaminid.In a further embodiment, the compound of formula (I) includes compoundsencompassed by formula (I) including formulae (IA), (IB), (IC), (ID),and (IE).

One embodiment of the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with clavulanate, meropenem, and SQ-109. Ina further embodiment, the compound of formula (I) includes compoundsencompassed by formula (I) including formulae (IA), (IB), (IC), (ID),and (IE).

In another embodiment, the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with one or more of thiacetazone,dirithromycin, roxithromycin, telithromycin, azithromycin,clarithromycin, erythromycin, amikacin, kanamycin, streptomycin, andlevofloxacin. In a further embodiment, the compound of formula (I)includes compounds encompassed by formula (I) including formulae (IA),(IB), (IC), (ID), and (IE).

In another embodiment, the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with pyrazinamide one or more ofthiacetazone, dirithromycin, roxithromycin, telithromycin, azithromycin,clarithromycin, erythromycin, amikacin, kanamycin, streptomycin, andlevofloxacin. In a further embodiment, the compound of formula (I)includes compounds encompassed by formula (I) including formulae (IA),(IB), (IC), (ID), and (IE).

In another embodiment, the invention provides methods and compositionscomprising the compound of formula (I), or a pharmaceutically acceptablesalt thereof, in combination with pyrazinamide one or more ofazithromycin, clarithromycin, erythromycin, amikacin, kanamycin,streptomycin, and levofloxacin. In a further embodiment, the compound offormula (I) includes compounds encompassed by formula (I) includingformulae (IA), (IB), (IC), (ID), and (IE).

The term “capuramycins” as used herein refers to a class of antibioticuseful for treating bacterial infections based on capuramycin.Capuramycin (general formula C₂₃H₃₁O₁₂N₅) is a nucleoside antibioticproduced by Streptomyces griseus 446-S3 active against streptococcuspneumoniae and Mycobacterium smegmatis ATCC 607 and has the followingstructure:

Examples of capuramycins suitable for the combinations of the presentapplication include capuramycin and the capuramycin analogue SQ-641.

As used herein, the term “aminocoumarin” refers to a class ofantibiotics that act by an inhibition of the DNA Gyrase enzyme involvedin the cell division in bacteria. They are derived from Streptomycesspecies. Examples of aminocoumarin antibiotics include novobiocin,cloroblocin, coumermycin, ferulobiocin, 3-chlorocoumarobiocin and8′-dechloro-3-chlorocoumarobiocin. Additional aminocoumarins suitablefor the combinations of the present invention include those described inLi, S. M. and Heide L. Curr. Med. Chem. 12:419-27 (2005) and inFriedman, M. et al. Biochem., 46:8462-71 (2007).

Isothiazoloquinolones suitable for the combinations of the presentinvention include ACH-710, ACH-702 (described by Pucci, M. J. et al. inAntimicrob. Agents Chemother., 55:2860-71 (2011)), and those disclosedin U.S. Application Publication No. 2012/0114601.

The term “benzothiazinones” as used herein refers to a class ofcompounds based on the structure:

or the structure:

useful for treating mycobacterial infection. Examples ofbenzothiazinones suitable for the combinations of the present inventioninclude BTZ 043, SKLB-TB37 and those disclosed in EP 2468746 andCN102276598,

Riminophenazine antibiotics suitable for the combinations of the presentinvention include clofazimine (B663) and B669. The term“diarylquinolone” as used herein refers to a class of compounds usefulfor treating mycobacterium and includes, but is not limited to,bedaquiline (TMC-207) and R207910. Bedaquiline has an IUPAC name of(1R,2S)-1-(6-Bromo-2-methoxy-3-quinolyl)-4-dimethylamino-2-(1-naphthyl)-1-phenyl-butan-2-oland may be synthesized according to the methods disclosed inWO2004/011436 and WO2006/125769.

The term “rifapentine” as used herein refers to an antibiotic useful fortreating bacterial infections. Rifapentine has an IUPAC name of(7S,9E,11S,12R,13S,14R,15R,16R,17S,18S,19E,21Z,26E)-26-{[(4-cyclopentylpiperazin-1-yl)amino]methylidene}-2,15,17,29-tetrahydroxy-1′-methoxy-3,7,12,14,16,18,22-heptamethyl-6,23,27-trioxo-8,30-dioxa-24-azatetracyclo[23.3.1.1^(4,7).0^(5,28)]triaconta-1(28),2,4,9,19,21,25(29)-heptaen-13-ylacetate and it may be purchased commercially (e.g., Sigma-Aldrich; Cat.No. R0533).

The term “rifampin” or “rifampicin” as used herein refers to anantibiotic useful for treating bacterial infections. Rifampin has anIUPAC name of:(7S,9E,11S,12R,13S,14R,15R,16R,17S,18S,19E,21Z)-2,15,17,27,29-pentahydroxy-1′-methoxy-3,7,12,14,16,18,22-heptamethyl-26-{(E)-[(4-methylpiperazin-1-yl)imino]methyl}-6,23-dioxo-8,30-dioxa-24-azatetracyclo[23.3.1.1^(4,7)0.0^(5,28)]triaconta-1(28),2,4,9,19,21,25(29),26-octaen-13-ylacetate and it may be purchased commercially (e.g., Fisher BioReagents(Cat No. BP2679-1) or Sigma-Aldrich (Cat. No. R3501)).

The following table provides structure and sources for some of theantibiotic compounds suitable for the combinations of the presentapplication.

TABLE 1 Compound (Name and/or Chemical Name and/or structure)Synthesis/Isolation/Commercial Availability Bedaquiline  

IUPAC name: (1R,2S)-1-(6-Bromo-2-mthoxy-3-quinolyl)-4-dimethylamino-2-(1-naphthyl)-1-phenyl- butan-2-ol  Synthesis: WO2004/011436 and process for chiral resolution in WO2006/125769. Rifapentine  

IUPAC Name: (7S,9E,11S,12R,13S,14R,15R,16R,17S,18S,19E,21Z,26E)-26-{[(4-cyclopentylpiperazin-1-yl)amino]methylidene}-2,15,17,29-tetrahydroxy-11-methoxy-3,7,12,14,16,18,22-heptamethyl-6,23,27- trioxo-8,30-dioxa-24-azatetracyclo[23.3.1.1^(4,7).0^(5,28)]triaconta-1(28),2,4,9,19,21,25(29)-heptaen-13-yl acetate  *Synthesis/Isolation/Commercial Availability (Sigma-Aldrich; Cat. No.R0533)

  Rifampin (Rifampicin or Rifadin): IUPAC name:(7S,9E,11S,12R,13S,14R,15R,16R,17S,18S,19E,21Z)-2,15,17,27,29-pentahydroxy-11-methoxy-3,7,12,14,16,18,22-heptamethyl-26-{(E)-[(4-methylpiperazin-1-yl)imino]methyl}-6,23-dioxo- 8,30-dioxa-24-azatetracyclo[23.3.1.1^(4,7).0^(5,28)]triaconta-1(28),2,4,9,19,21,25(29),26-octaen-13-yl acetate.  Synthesis/Isolation/Commercial Availability: can be purchased fromFisher BioReagents (Cat No. BP2679-1) or Sigma-Aldrich (Cat. No. R3501)

  Pyrazinamide *IUPAC Name: pyrazine-2-carboxamide  Synthesis/Isolation/Commercial Availability: can be purchased fromseveral U.S. sources (Sigma-Aldrich Cat. No. P1736) Isoniazid: (alsoabbreviated as INH; generic)  

*IUPAC Name: isonicotinohydrazide   *Synthesis/Isolation/CommercialAvailability: can be purchased from Sigma Aldrich (Cat. No. I3377)Moxifloxacin  

*IUPAC name: 1-cyclopropyl-7-[(1S,6S)-2,8- diazabicyclo[4.3.0]non-8-yl]-6-fluoro-8-methoxy-4-oxo- quinoline-3-carboxylic acid **Synthesis/Isolation/Commercial Availability: can be purchased fromSigma Aldrich (Cat. No. 32477) Gatifloxacin (Gatiflo, Tequin, Zymar,BMS, Kyorin)  

*IUPAC name: 1-cyclopropyl-6-fluoro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxo-quinoline-3- carboxylic acid  *Synthesis/Isolation/Commercial Availability: can be purchased fromSigma Aldrich (Cat. No. G7298) Linezolid  

*IUPAC Name: (S)-N-({3-[3-fluoro-4-(morpholin-4-yl)phenyl]-2-oxo-1,3-oxazolidin-5- yl}methyl)acetamide  Synthesis/Isolation/Commercial Availability: can be purchased from SigmaAldrich (Cat. No. PZ0014) Sutezolid (PNU-100480, Pfizer):  

Synthesis/Isolation/Commercial Availability: can be purchased from AxonMedChem BV, Postbus 770 Groningen, 9700 AT Netherlands (Cat. No. Axon1762) Posizolid (Astra Zeneca)  

IUPAC Name: (5R)-3-[4-[1-[(2S)-2,3-Dihydroxypropanoyl]-3,6-dihydro-2H-pyridin-4-yl]-3,5-difluorophenyl]-5-(1,2-oxazol-3-yloxymethyl)- 1,3-oxazolidin-2-one  Synthesis/Isolation/Commercial Availability: can be purchased from BOCSciences, 45-16 Ramsey Road, Shirley, NY 11967 (Cat. No. 252260-02-9)AZD-5847 *Synthesis/Isolation: Process for preparing the phosphate esterprodrug WO0140236. PA 824  

*IUPAC name: (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H- imidazo[2,1-b][1,3]oxazine  *Synthesis/Isolation/Commercial Availability: can be purchased fromBOC Sciences, 45-16 Ramsey Road, Shirley, NY 11967 (Cat. No.187235-37-6) Ethambutol  

IUPAC name: (2S,2′S)-2,2′-(Ethane-1,2- diyldiimino)dibutan-1-ol  *Synthesis/Isolation/Commercial Availability: can be purchased fromSigma Aldrich (Cat. No. E4630) SQ-109: (Sequella)  

*Synthesis/Isolation/Commercial Availability: can be purchased fromAurora Fire Chemicals LLC, 7929 Silverton Ave., San Diego, CA 92126(Cat. No. K06.990.223); also see U.S. Pat. No. 6,951,961) SQ-609(Sequella):  

*IUPAC Name: 1-{[1-(Adamantan-1-ylmethyl)-4-piperidinyl]methyl}-4-piperidinol   *Synthesis/Isolation/CommercialAvailability: WO03096987; also Bioorganic and Med Chem Letters 21 (18),pp 5353-5357 (2011) and Bioorganic and Med Chem Letters 20 (1), pp.201-205 (2010).

*Synthesis/Isolation/Commercial Availability: WO2009136965; alsoBioorganic and Med Chem Letters 21 (18), pp. 5353-5357 (2011) Delaminid(OPC-67683, Otsuka)  

Chemical name: (2R)-2-methyl-6-nitro-2-[(4-{4-[4-(trifluoromethoxy)phenoxy]piperidin-1-yl}phenoxy)methyl]-2,3-dihydroimidazo[2,1- b]oxazole  *Synthesis/Isolation/Commercial Availability: JP2005330266;WO20044033463; WO2004035547 (process app) also J. Med Chem. 49 (26), pp.7854- 7860 (2006) Clofazimine (Lamprine)  

IUPAC name: N,5-bis(4-chlorophenyl)-3-(propan-2-ylimino)-3,5-dihydrophenazin-2-amine   *Synthesis/Isolation/CommercialAvailability: can be purchased from Sigma Aldrich (Cat. No. C8895)Dapsone (Aczone)  

*IUPAC name: 4-[(4-aminobenzene)sulfonyl]aniline  *Synthesis/Isolation/Commercial Availability: can be purchased fromSigma Aldrich (Cat. No. A74807) Rifafour A combo tablet containing the 4first line TB agents of Rifampicin (150 mg) + Isoniazid (75 mg) +Pyrazinamide (400 mg) + Ethambutol (275 mg). BTZ 043  

*Synthesis/Isolation/Commercial Availability: can be purchased from BOCSciences, 45-16 Ramsey Road, Shirley, NY 11967 (Cat. No. 1161233-85-7)Capreomycin  

*Synthesis/Isolation/Commercial Availability: can be purchased fromSigma Aldrich (Cat. No. C4142) Cycloserine  

Synthesis/Isolation/Commercial Availability: can be purchased from SigmaAldrich (Cat. No. C7005 for DL-Cycloserine; C1159 for L-Cycloserine;C6680 for D-Cycloserine) Ethionamide  

Synthesis/Isolation/Commercial Availability: can be purchased from SigmaAldrich (Cat. No. E6005) DC-159a  

*Synthesis/Isolation/Commercial Availability: WO2007111023 (in Japanese;Daiichi Pharmaceutical Co.); also Bioorganic and Med Chem Letters 21(18), pp. 5353-5357 (2011) Prothionamide  

*Synthesis/Isolation/Commercial Availability: can be purchased from BOCSciences, 45-16 Ramsey Road, Shirley, NY 11967 (Cat. No. 14222-60-7)4-aminosalicyclic acid  

*Synthesis/Isolation/Commercial Availability: can be purchased from AlfaAesar, 26 Parkridge Rd., Ward Hill, MA 01835 (Cat. No. B23289) Rifalazil 

*Synthesis/Isolation/Commercial Availability: can be purchased from BOCSciences, 45-16 Ramsey Road, Shirley, NY 11967 (Cat. No. 129791-92-0)CPZEN45 (Caprazamycin analogue)  

*Synthesis/Isolation/Commercial Availability: WO2010038874 andWO2008020560 (both in Japanese); also Bioorganic and Med Chem Letters 21(18), pp. 5353-5357 (2011) ACH-710 (isothiazoloquinolone)  

  FIG. 1/Chemical structure of ACH-702. *Synthesis/Isolation/CommercialAvailability: WO200821491 and WO2007014308 (both in Japanese); alsoBioorganic and Med Chem Letters 21 (18), pp. 5353-5357 (2011) Meropenem(beta lactam class and belongs to the carbapenem subgroup)  

*Synthesis/Isolation/Commercial Availability: can be purchased fromSigma Aldrich (Cat. No. M2574) Clavulanate (Clavulanate Potassium) (Betalactamase inhibitors) Clavulanic acid  

*Synthesis/Isolation/Commercial Availability: can be purchased fromSigma Aldrich (Cat. No. 33454) Thioridiazine  

Synthesis/Isolation/Commercial Availability: can be purchased from SigmaAldrich (Cat. No. T9025) Q201 (Imidazopyridine) in preclinicaldevelopment by Qurient Therapeutics) Thiacetazone  

Approved drug Levofloxacin  

*Commercial Availability: can be purchased from Sigma Aldrich (Cat. No.28266) SKLB-TB37  

*Synthesis/Isolation: CN102276598A Azithromycin  

*Commercial Availability: can be purchased from\ Sigma Aldrich (Cat. No.PZ0007) Clarithromycin  

*Commercial Availability: can be purchased from Sigma Aldrich (Cat. No.C9742) Erythromycin  

*Commercial Availability: can be purchased from Sigma Aldrich (Cat. No.E6376) Dirithromycin  

*Commercial Availability: can be purchased from Sigma Aldrich (Cat. No.D4065) Roxithromycin  

*Commercial Availability: can be purchased from Sigma Aldrich (Cat. No.R4393) Telithromycin  

*Approved Drug

For the compound of formula (I), dosage levels of between about 0.01 andabout 100 mg/kg body weight per day, preferably between 0.5 and about 75mg/kg body weight per day and most preferably between about 1 and 50mg/kg body weight per day of the active ingredient compound are usefulin a monotherapy for the prevention and treatment of bacterialinfections.

Typically, the pharmaceutical compositions of this invention will beadministered from about 1 to 5 times per day or alternatively, as acontinuous infusion. Alternatively, the compositions of the presentinvention may be administered in a pulsatile formulation. Suchadministration can be used as a chronic or acute therapy. The amount ofactive ingredient that may be combined with the carrier materials toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration A typical preparation willcontain from about 5% to about 95% active compound (w/w). Preferably,such preparations contain from about 20% to about 80% active compound.

When the compositions of this invention comprise a combination of acompound of formula (I) and one or more additional therapeutic orprophylactic agents, both the compound and the additional agent shouldbe present at dosage levels of between about 10% to 80% of the dosagenormally administered in a monotherapy regime.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence or disease symptoms.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, sex, diet, time of administration, rateof excretion, drug combination, the severity and course of the disease,and the patient's disposition to the disease and the judgment of thetreating physician.

According to another embodiment, the invention provides methods fortreating or preventing a bacterial infection, or disease state,comprising the step of administering to a patient any compound,pharmaceutical composition, or combination described herein. The term“patient”, as used herein, means an animal, preferably a mammal, andmost preferably a human.

The U.S. Department of Health and Human Services Centers for DiseaseControl and Prevention have published recommendations for the treatmentof tuberculosis. Centers for Disease Control and Prevention. Treatmentof Tuberculosis, American Thoracic Society, CDC, and Infectious DiseasesSociety of America. MMWR 2003; 52(No. RR-11):1-80, incorporated byreference herein as if fully set forth. In these recommendations aredisclosed various first-line and second-line drugs used to treat TB aswell as recommended treatment regimens including combinations of knowncompounds, the recommended interval and dosages and range of total dosesfor both adults and children. In addition, treatment algorithms for TBand active, culture-negative pulmonary TB and inactive TB are providedas well as management of relapse, treatment failure and drug resistance.For the purposes of this invention, a compound of formula (I) may beadded to the specific treatment regimens set forth therein orsubstituted for one of the components listed in the treatment regimens,either as a first-line or second-line drug.

The recommended treatment regimens are, in large part, based on evidencefrom clinical trials and are rated on the basis of a system developed bythe United States Public Health Service (USPHS) and the InfectionDiseases Society of America (IDSA). The rating system includes a letter(A, B, C, D, or E) that indicates the strength of the recommendation anda roman numeral (I, II, or III) that indicates the quality of evidencesupporting the recommendation (Table 2).

There are four recommended regimens for treating patients with TB causedby drug-susceptible organisms as set forth in the 2003 guidancedocument. Each regimen has an initial phase of 2 months followed by achoice of several options for the continuation phase of either 4 or 7months. The recommended regimens together with the number of dosesspecified by the regimen are described in Table 3. The initial phasesare denoted by a number (1, 2, 3, or 4) and the continuation phases thatrelate to the initial phase are denoted by the number plus a letterdesignation (a, b, or c). Drug doses are shown in Tables 4, 5, and 6.

TABLE 2a Infectious Diseases Society of America/United States PublicHealth Service rating system for the strength of treatmentrecommendations based on quality of evidence Strength of therecommendation A. Preferred; should generally be offered B. Alternative;acceptable to offer C. Offer when preferred or alternative regimenscannot be given D. Should generally not be offered E. Should never beoffered Quality of evidence supporting the recommendation I. At leastone properly randomized trial with clinical end points II. Clinicaltrials that either are not randomized or were conducted in otherpopulations III. Expert opinion

TABLE 2b Drug regimens for culture-positive pulmonary tuberculosiscaused by drug-susceptible organisms Initial phrase Continuation phaseRange of total Rating* Interval and doses^(‡) Interval and doses^(‡§)doses (minimal (evidence)^(†) Regimen Drugs (minimal duration) RegimenDrugs (minimal duration) duration) HIV⁻ HIV⁺ 1 INH Seven days per weekfor 56 doses 1a INH/RIF Seven days per week for 126 182-130 (26 wk) A(I) A (II) RIF (8 wk) or 5 d/wk for 40 doses doses (18 wk) or 5 d/wk for90 PZA (8 wk)^(¶) doses (18 wk)^(¶) EMB 1b INH/RIF Twice weekly for 36doses (18 wk)  92-76 (26 wk) A (I) A (II)^(#) 1c** INH/RPT Once weeklyfor 18 doses (18 wk)  74-58 (26 wk) B (I) E (I) 2 INH Seven days perweek for 14 doses 2a INH/RIF Twice weekly for 36 doses (18 wk)  62-58(26 wk) A (II) B (II)^(#) RIF (2 wk), then twice weekly for 12 2b**INH/RPT Once weekly for 18 doses (18 wk)  44-40 (26 wk) B (I) E (I) PZAdoses (6 wk) or 5 d/wk for 10 EMB doses (2 wk),^(¶) then twice weeklyfor 12 doses (6 wk) 3 INH Three times weekly for 24 doses 3a INH/RIFThree times weekly for 54 doses  78 (26 wk) B (I) B (II) RIF (8 wk) (18wk) PZA EMB 4 INH Seven days per week for 56 doses 4a INH/RIF Seven daysper week for 217 273-195 (39 wk) C (I) C (II) RIF (8 wk) or 5 d/wk for40 doses doses (31 wk) or 5 d/wk for 155 EMB (8 wk)^(¶) 4b INH/RIF doses(31 wk)^(¶) 118-102 (39 wk) C (I) C (II) Twice weekly for 62 doses (31wk) Definition of abbreviations: EMB = Ethambutol; iNH = isoniazid; PZA= pyrazinamide; RIF = rifampin; RPT = rifapentine *Definitions ofevidence ratings: A = preferred; B = acceptable alternative; C = offerwhen A and B cannot be given; E = should never be given. ^(†)Definitionof evidence ratings: I = randomized clinical trial; II = data fromclinical trials that were not randomized or were conducted in otherpopulations; III = expert opinion. ^(‡)When DOT is used, drugs may begiven 5 days/week and the necessary number of doses adjustedaccordingly. Although there are no studies that compare five with sevendaily doses, extensive experience indicates this would be an effectivepractice. ^(§)Patients with cavitation on initial chest radiograph andpositive cultures at completion of 2 months of therapy should receive a7-month (31 week; either 217 doses [daily] or 62 doses [twice weekly])continuation phase. ^(¶)Five-day-a-week administration is always givenby DOT. Rating for 5 day/week regimens is AIII ^(#)Not recommended forHIV-infected patients with CD4⁺ cell counts <100 cells/μl. **Options 1cand 2b should be used only in HIV-negative patients who have negativesputum smears at the time of completion of 2 months of therapy and whodo not have cavitation on initial chest radiograph (see text). Forpatients started on this regimen and found to have a positive culturefrom the 2-month specimen, treatment should be extended an extra 3months.

TABLE 3 Doses of antituberculosis drugs for adults and children DosesDrug Preparation Adults/children Daily 11×/wk 2×/wk 3×/wk First-linedrugs Isoniazid Tablets (50 mg, 100 mg, Adults (max.) 5 mg/kg (300 mg)15 mg/kg (900 mg) 15 mg/kg (900 mg) 15 mg/kg (900 mg) 300 mg); elixir(50 mg/ Children (max.) 10-15 mg/kg (300 mg) — 20-30 mg/kg — 5 ml);aqueous solution (900 mg) (100 mg/ml) for intravenous or intramuscularinjection Rifampin Capsule (150 mg, 300 Adults^(‡) (max.) 10 mg/kg (600mg) — 10 mg/kg (600 mg) 10 mg/kg (600 mg) mg); powder may be Children(max.) 10-20 mg/kg (600 mg) — 10-20 mg/kg — suspended for oral (600 mg)administration, aqueous solution for intravenous injection RifabutinCapsule (150 mg) Adults^(‡) (max.) 5 mg/kg (300 mg) — 6 mg/kg (300 mg) 5mg/kg (300 mg) Children Appropriate dosing for Appropriate dosingAppropriate dosing Appropriate dosing children is unknown for childrenis for children is for children is unknown unknown unknown RifapentineTablet (150 mg, Adults — 10 mg/kg (continua- — — film coated) tion phase(600 mg) Children The drug is not approved The drug is not The drug isnot The drug is not for use in children approved for use in approved foruse in approved for use in children children children PyrazinamideTablet (500 mg, scored) Adults See Table 4 — See Table 4 See Table 4Children (max.) 15-30 mg/kg (2.0 g) — 50 mg/kg (2 g) — Ethambutol Tablet(100 mg, Adults See Table 5 — See Table 5 See Table 5 400 mg)Children^(§) (max.) 15-20 mg/kg daily — 50 mg/kg (2.5 g) — (1.0 g)Second-line drugs Cyclosenine Capsule (250 mg) Adults (max.) 10-15mg/kg/d (1.0 g in There are no data to There are no data to There are nodata to two doses), usually support intermittant support intermittantsupport intermittant 500-750 mg/d in two admisistration administrationadministration doses^(¶) Children (max.) 10-15 mg/kg/d (1.0 g/d) — — —Ethionamide Tablet (250 mg) Adults* (max.) 15-20 mg/kg/d (1.0 g/d),There are no data to There are no data to There are no data to usually500-750 mg/d support intermittent support intermittent supportintermittent in a single daily dose or administration administrationadministration two divided doses^(#) Children (max.) 15-20 mg/kg/d (1.0g/d) There are no data to There are no data to There are no data tosupport intermittent support intermittent support intermittentadministration administration administration Streptomycin Aqueoussolution Adults (max.) — ** ** ** (1-g vials) for intrave- Children(max.) 20-40 mg/kg/d (1 g) — 20 mg/kg — nous or intramuscularadministration Amikacin/ Aqueous solution Adults (max.) — ** ** **kanamycin (500-mg and 1-g vials) Children (max.) 15-30 mg/kg/d (1 g) —15-30 mg/kg — for intravenous or intra- intravenous or muscularadministration intramuscular as a single daily dose Capreomycin Aqueoussulution Adults (max.) — ** ** ** (1-g vials) for intrave- Children(max.) 15-30 mg/kg/d (1 g) as a — 15-30 mg/kg — nous or intramuscularsingle daily dose administration p- Granules (4-g packets) Adults 8-12g/d in two or three There are no data to There are no data to There areno data to Amino- can be mixed with food; doses support intermittentsupport intermittent support intermittent salicylic acid tablets (500mg) are still administration administration administration (PAS)available in some Children 200-300 mg/kg/d in two There are no data toThere are no data to There are no data to countries, but not in the tofour divided doses suppport intermittent support intermittent supportintermittent United States; a solution (10 g) administrationadministration administration for intravenous admini- stration isavailable in Europe Levofloxacin Tablets (250 mg, 500 Adults 500-1,000mg daily There are no data to There are no data to There are no data tomg, 750 mg); aqueous support intermittent support intermittent supportintermittent solution (500-mg vials) administration administrationadministration for intravenous injec- Children †† †† †† †† tionMoxifloxacin Tablets (400 mg); Adults 400 mg daily There are no data toThere are no data to There are no data to aqueous solution supportintermittent support intermittent support intermittent (400 mg/250 ml)for administration administration administration intravenous injectionChildren ‡‡ ‡‡ ‡‡ ‡‡ Gatifloxacin Tablets (400 mg); Adults 400 mg dailyThere are no data to There are no data to There are no data to aqueoussolution support intermittent support intermittent support intermittent(200 mg/20 ml; 400 administration administration administration mg/40ml) for Children §§ §§ §§ §§ intravenous injection *Dose per weight isbased on ideal body weight. Children weighing more than 40 kg should bedosed as adults. ^(†)For purpose of this document adult dosing begins atage 15 years. ^(‡)Dose may need to be adjusted when there is concomitantuse of protease inhibitors or nonnucleoside reverse transcriptaseinhibitors. ^(§)The drug can likely be used safely in older children butshould be used with caution in children less than 5 years of age, inwhom visual acuity cannot be monitored. In younger children EMB at thedose of 15 mg/kg per day can be used if there is suspected or provenresistance to INH or RIF. ^(¶)It should be noted that, although this isthe dose recommended generally, most clinicians with experience usingcycloserine indicate that it is unusual for patients to be able totolerate this amount. Serum concentration measurements are often usefulin determining the optimal dose for a given patient. The single dose canbe given at bedtime or with the main meal. **Dose: 15 mg/kg per day (1g), and 10 mg/kg in persons more than 59 years of age (750 mg). Usualdose 750-1,000 mg administered intramuscularly or intravenously, givenas a single dose 5-7 days/week and reduced to two or three times perweek after the first 2-4 months or after culture conversion, dependingon the efficacy of the other drugs in the regimen. ††The long-term (morethan several weeks) use of levofloxacin in children and adolescents hasnot been approved because of concerns about effects on bone andcartilage growth. However, most experts agree that the drug should beconsidered for children with tuberculosis caused by organisms resistantto both INH and RIF. The optimal dose is not known. ‡‡The long-term(more than several weeks) use of moxifloxacin in children andadolescents has not been approved because of concerns about effects onbone and cartilage growth. The optimal dose is not known. §§Thelong-term (more than several weeks) use of gatifloxacin in children andadolescents has not been approved because of concerns about effects onbone and cartilage grwoth. The optimal dose is not known.

TABLE 4 Suggested pyrazinamide doses, using whole tablets, for adultsweighing 40-90 kg Weight (kg)* 40-55 56-75 76-90 Daily, mg 1,000(18.2-25.0) 1,500 (20.0-26.8) 2,000^(†) (22.2-26.3) (mg/kg) Thrice 1,500(27.3-37.5) 2,500 (33.3-44.6) 3,000^(†) (33.3-39.5) weekly, mg (mg/kg)Twice 2,000 (36.4-50.0) 3,000 (40.0-53.6) 4,000^(†) (44.4-52.8) weekly,mg (mg/kg) *Based on estimated lean body weight. ^(†)Maximum doseregardless of weight.

TABLE 5 Suggested ethambutol doses, using whole tablets, for adultsweighing 40-90 kg Weight (kg)* 40-55 56-75 76-90 Daily, mg  800(14.5-20.0) 1,200 (16.0-21.4) 1,600^(†) (17.8-21.1) (mg/kg) Thrice 1,200(21.8-30.0) 2,000 (26.7-35.7) 2,400^(†) (26.7-31.6) weekly, mg (mg/kg)Twice 2,000 (36.4-50.0) 2,800 (37.3-50.0) 4,000^(†) (44.4-52.6) weekly,mg (mg/kg) *Based on estimated lean body weight. ^(†)Maximum doseregardless of weight.

The term “prodrug” refers to compounds which are drug precursors which,following administration and absorption, release the drug in vivo viasome metabolic process. In general, a prodrug possesses less biologicalactivity than its parent drug. A prodrug may also improve the physicalproperties of the parent drug and/or it may also improve overall drugefficacy, for example through the reduction of toxicity and unwantedeffects of a drug by controlling its absorption, blood levels, metabolicdistribution and cellular uptake.

The term “parent compound” or “parent drug” refers to the biologicallyactive entity that is released via enzymatic action of a metabolic or acatabolic process, or via a chemical process following administration ofthe prodrug. The parent compound may also be the starting material forthe preparation of its corresponding prodrug.

The monovalent cations defined by M⁺ include ammonium, alkali metal ionssuch as sodium, lithium and potassium ions, dicyclohexylamine ion, andN-methyl-D-glucamine ion. The divalent cations defined by D²⁺ include,alkaline earth metal ions such as aluminum, calcium and magnesium ions.Also included are amino acid cations such as ions of arginine, lysine,ornithine, and so forth. If M⁺ is a monovalent cation, it is recognizedthat if the definition 2M⁺ is present, each of M⁺ may be the same ordifferent. In addition, it is similarly recognized that if thedefinition 2M⁺ is present, a divalent cation D²⁺ may instead be present.Also, the basic nitrogen-containing groups may be quaternized with suchagents as: lower alkyl halides, such as methyl, ethyl, propyl, and butylchloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl,dibutyl; diamyl sulfates; long chain halides such as decyl, lauryl,myristyl and stearyl chlorides, bromides and iodides; aralkyl halideslike benzyl bromide and others.

Various embodiments of the invention, include compounds or salts offormula (IB) as set forth below:

-   -   (1) compounds wherein X is    -   (a) —PO(OH)O⁻M⁺;    -   (b) —PO(O⁻)₂.2M⁺; or    -   (c) —PO(O⁻)₂.D²⁺;    -   (2) compounds wherein M⁺ is    -   (a) Li⁺, Na⁺, K⁺, N-methyl-D-glucamine, or N(R⁹)₄ ⁺; or    -   (b) Na⁺;    -   (c) each R⁹ is independently hydrogen or a C₁-C₄ alkyl group;    -   (3) compounds wherein D²⁺ is    -   (a) Mg²⁺, Ca²⁺, and Ba²⁺; or    -   (b) Ca²⁺;    -   (4) the compound        (R)-2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-yl        phosphate; and    -   (5) the compound disodium        (R)-2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-yl        phosphate.

It is understood that various alternative embodiments of the compoundsor salts of formula (IB) can be selected by requiring one or more of thealternate embodiments listed in (1) through (3) above. For example,further embodiments of the invention can be obtained by combining (1)(a)and (2)(a); (1)(a) and (2)(b); (1)(c) and (3)(a); (1)(c) and (3)(b);(1)(b) and (2)(a); (1)(b) and (2)(b); and the like.

The prodrugs of the present invention are characterized by unexpectedlyhigh aqueous solubility. This solubility facilitates administration ofhigher doses of the prodrug, resulting in a greater drug load per unitdosage.

A “pharmaceutically acceptable derivative or prodrug” means anypharmaceutically acceptable salt, ester, salt of an ester or otherderivative of a compound of this invention which, upon administration toa recipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof. Particularly favored derivatives or prodrugs are thosethat increase the bioavailability of the compounds of this inventionwhen such compounds are administered to a mammal (e.g., by allowing anorally administered compound to be more readily absorbed into the blood)or which enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) relative to the parentspecies.

Pharmaceutically acceptable prodrugs of the compounds of this inventioninclude, without limitation, esters, amino acid esters, phosphateesters, metal salts and sulfonate esters.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate,pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,propionate, salicylate, succinate, sulfate, tartrate, thiocyanate,tosylate and undecanoate. Other acids, such as oxalic, while not inthemselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium andN⁺(C₁₋₄ alkyl)₄ salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization.

Pharmaceutical compositions of this invention comprise a compound offormula (I) or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier. Such compositions may optionallycomprise an additional therapeutic agent. Such agents include, but arenot limited to, an antibiotic, an anti-inflammatory agent, a matrixmetalloprotease inhibitor, a lipoxygenase inhibitor, a cytokineantagonist, an immunosuppressant, an anti-cancer agent, an anti-viralagent, a cytokine, a growth factor, an immunomodulator, a prostaglandinor an anti-vascular hyperproliferation compound.

The term “pharmaceutically acceptable carrier” refers to a non-toxiccarrier that may be administered to a patient, together with a compoundof this invention, and which does not destroy the pharmacologicalactivity thereof.

Pharmaceutically acceptable carriers that may be used in thepharmaceutical compositions of this invention include, but are notlimited to, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat and self-emulsifying drug delivery systems (SEDDS) such asalpha-tocopherol, polyethyleneglycol 1000 succinate, or other similarpolymeric delivery matrices.

The term “pharmaceutically effective amount” or “therapeuticallyeffective amount” refers to an amount effective in treating orameliorating a bacterial infection in a patient. The term“prophylactically effective amount” refers to an amount effective inpreventing or substantially lessening a bacterial infection in apatient.

The following definitions describe terms and abbreviations used herein:

-   Ac acetyl-   Bu butyl-   Et ethyl-   Ph phenyl-   Me methyl-   THF tetrahydrofuran-   DCM dichloromethane-   CH₂Cl₂ dichloromethane-   EtOAc ethyl acetate-   CH₃CN acetonitrile-   EtOH ethanol-   Et₂O diethyl ether-   MeOH methanol-   MTBE methyl tert-butyl ether-   DMF N,N-dimethylformamide-   DMA N,N-dimethylacetamide-   DMSO dimethyl sulfoxide-   HOAc acetic acid-   TEA triethylamine-   TFA trifluoroacetic acid-   TFAA trifluoroacetic anhydride-   Et₃N triethylamine-   DIPEA diisopropylethylamine-   DIEA diisopropylethylamine-   K₂CO₃ potassium carbonate-   Na₂CO₃ sodium carbonate-   Na₂S₂O₃ sodium thiosulfate-   Cs₂CO₃ cesium carbonate-   NaHCO₃ sodium bicarbonate-   NaOH sodium hydroxide-   Na₂SO₄ sodium sulfate-   MgSO₄, magnesium sulfate-   K₃PO₄ potassium phosphate-   NH₄Cl ammonium chloride-   LC/MS liquid chromatography/mass spectra-   GCMS gas chromatography mass spectra-   HPLC high performance liquid chromatography-   GC gas chromatography-   LC liquid chromatography-   IC ion chromatography-   IM intramuscular-   CFU/cfu colony forming units-   MIC minimum inhibitory concentration-   Hr or h hours-   atm atmospheres-   rt or RT room temperature-   TLC thin layer chromatography-   HCl hydrochloric acid-   H₂O water-   EtNCO ethyl isocyanate-   Pd/C palladium on carbon-   NaOAc sodium acetate-   H₂SO₄ sulfuric acid-   N₂ nitrogen gas-   H₂ hydrogen gas-   n-BuLi n-butyl lithium-   DI de-ionized-   Pd(OAc)₂ palladium(II)acetate-   PPh₃ triphenylphosphine-   i-PrOH isopropyl alcohol-   NBS N-bromosuccinimide-   Pd[(Ph₃)P]₄ tetrakis(triphenylphosphine)palladium(0)-   PTFE polytetrafluoroethylene-   rpm revolutions per minute-   SM starting material-   Equiv. equivalents-   ¹H-NMR proton nuclear magnetic resonance-   HPMCAS hydroxypropylmethylcellulose acetate-   PVP polyvinylpyrrolidone-   EDTA ethylenediaminetetraacetic acid-   K2EDTA dibasic potassium ethylenediaminetetraacetate-   mCPBA meta-chloroperoxybenzoic acid-   aq aqueous-   Boc₂O di-tert-butyl dicarbonate-   DMAP N,N-dimethylaminopyridine-   mL milliliters-   L liters-   mol moles-   g grams-   LCMS liquid chromatography-mass spectrometry-   MHz megahertz-   CDCl₃ deuterochloroform-   NEt₃ triethylamine-   mmol millimoles-   psi pounds per square inch-   iPrOH isopropylalcohol-   ppm parts per million-   NH₄NO₃ ammonium nitrate-   Hz hertz-   Pd(dppf)Cl_(2 [)1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)-   L liters-   MeOD deutero-methanol-   CD₃OD deutero-methanol-   ee enantiomeric excess-   min minutes-   Bn benzyl-   RBF round-bottom flask-   MeCN acetonitrile-   PES polyethersulfone-   mm millimeters-   μm micrometers-   M molar-   N normal-   Boc tert-butoxycarbonyl-   ESMS electrospray mass spectrometry-   CV column volume-   D₂O deuterium oxide-   NH₃ ammonia-   OBD optimum bed density-   mg milligrams-   CLSI Clinical and Laboratory Standards Institute-   ATCC American Type Culture Collection-   MHII Mueller Hinton II-   μm microliters-   WT wild type-   CGSC Coli Genetic Stock Center-   MS mass spectrometry-   IS internal standard-   APCI atmospheric pressure chemical ionization-   MRM multiple reaction monitoring-   m/z mass-to-charge ratio-   LLOQ lower limit of quantitation-   ng nanograms-   UV ultraviolet-   SD standard deviation-   % CV coefficient of variation-   PO perioral-   MC microcrystalline cellulose-   EDTA ethylenediaminetetraacetic acid or ethylenediaminetetraacetate-   PK pharmacokinetic-   PBS phosphate buffer saline-   IV intravenous-   D5W 5% dextrose in water solution-   HPMC-AS hydroxypropyl methylcellulose acetyl succinate-   PVP polyvinylprrolidone-   CAPT captisol-   ATP adenosine triphosphate-   ADP adenosine diphosphate-   NADH nicotinamide adenine dinucleotide (reduced form)-   NAD+ nicotinamide adenine dinucleotide (oxidized form)-   TRIS tris(hydroxymethyl)aminomethane-   mM millimolar-   MgCl₂ magnesium chloride-   KCl potassium chloride-   μM micromolar-   DTT dithiothreitol-   nM nanomolar-   dissociation constant-   IC₅₀ half maximal inhibitory concentration-   μg micrograms-   BSA bovine serum albumin-   LDH lactate dehydrogenase-   PVDF polyvinylidene fluoride-   PBS phosphate buffered saline-   BSL3 Biosafety Level 3-   AcN acetonitrile-   V_(MAX) the maximum initial velocity or rate of a reaction

EXAMPLE 1 Preparation of 2-(2-nitrophenyl)-2,5-dihydrofuran and2-(2-nitrophenyl)-2,3-dihydrofuran (3a&3b)

Mixed 1-bromo-2-nitro-benzene (1) (600 g, 99%, 2.941 mol, Alfa AesarA11686), 1,3-bis(diphenylphosphino)propane (62.50 g, 97%, 147.0 mmol,Alfa Aesar A12931), 1,4-dioxane (2.970 L, Sigma-Aldrich 360481),potassium carbonate (812.9 g, 5.882 mol, JT-Baker 301201), and2,3-dihydrofuran (2) (1.041 kg, 99%, 1.124 L, 14.70 mol, Aldrich200018). A stream of nitrogen was bubbled through the stirring mixturefor 4 hrs, followed by addition of palladium (II) acetate (16.51 g,73.52 mmol, Strem 461780) and continuation of deoxygenation for another10 minutes. The reaction mixture was stirred at reflux under nitrogenovernight (NMR of a worked-up aliquot showed complete consumption ofarylbromide). It was allowed to cool, diluted with hexane (1 L),filtered through a short plug of Florisil® (500 g, −200 mesh), andeluted with EtOAc. The filtrate was concentrated under reduced pressure(2-(2-nitrophenyl)-2,3-dihydrofuran is volatile under high vacuum andmay be somewhat unstable at room temperature) giving a mixture of (3a)and (3b) as a dark brown oil (654.0 g). The crude material was stored inthe refrigerator and carried forward without further purification.

EXAMPLE 2 Preparation of 2-tetrahydrofuran-2-yl-aniline (4)

Placed 5% palladium on carbon (16.3 g, 50% wet, 3.83 mmol, Aldrich330116) in a Parr bottle under nitrogen, followed by MeOH (100 mL,JT-Baker 909333). Added the crude mixture of2-(2-nitrophenyl)-2,5-dihydrofuran and2-(2-nitrophenyl)-2,3-dihydrofuran (3a&3b)) (163 g) dissolved in MeOH(389 mL), followed by NEt₃ (237.6 mL, 1.705 mol, Sigma-Aldrich 471283).Placed the vessel on a Parr shaker and saturated with H₂. Added 30 psiH₂ and shook until consumption complete (LCMS and NMR showed completereaction). The reaction mixture was purged with nitrogen, filteredthrough Celite™ and rinsed with EtOAc. The filtrate was concentrated ona rotary evaporator giving a brown oil. Repeated the reaction three moretimes on the same scale and the batches were combined for purification.The crude product was vacuum distilled (ca. 15 ton) collecting thedistillate at 108-129° C. to give (4) as a clear faint yellow oil (427.9g, average yield was 84%; 98% GCMS purity). LCMS (C18 column elutingwith 10-90% CH₃CN/water gradient over 5 minutes with formic acidmodifier) M+1: 163.95 (1.46 min). ¹H NMR (300 MHz, CDCl₃). δ 7.15-7.04(m, 2H), 6.77-6.62 (m, 2H), 4.85-4.77 (m, 1H), 4.18 (s, 2H), 4.12-4.02(m, 1H), 3.94-3.85 (m, 1H), 2.25-1.95 (m, 4H) ppm.

EXAMPLE 2a Preparation of (R)-2-(tetrahydrofuran-2-yl)aniline (4a)

Dissolved 33 g of compound (4) into MeOH (265 ml) which resulted in aconcentration of approximately 125 mg/ml. The mixture was filteredthrough a 0.2 micron membrane filter then chromatographed on aChiralPak® IC column (30 mm×150 mm, column temp 35° C., ChiralTechnologies) at 100 bar using a Berger multigram supercritical fluidchromatographic system. Mobile phase was (90:10) CO₂:CH₃OH eluting at350 ml/min with UV monitoring at 220 nanometers. Obtained 15.64 g ofdesired product (4a) as a green oil. Analytical SFC ([90:10] CO₂:CH₃OH,at 5 ml/min on a ChiralPak IC column (4.6×100 mm) held at 35° C. and runat 100 bar pressure with UV monitoring at 220 nm) showed 95.5% ee with95% overall purity.

EXAMPLE 3 Preparation of 4-bromo-2-tetrahydrofuran-2-yl-aniline (5)

To a stirring solution of 2-tetrahydrofuran-2-yl-aniline (4) (53.45 g,327.5 mmol) in methyl tert-butyl ether (MTBE, 641.4 mL) and acetonitrile(213.8 mL) cooled to 2° C. was added N-bromosuccinimide (58.88 g, 99%,327.5 mmol, Aldrich B81255) in 4 portions maintaining internaltemperature below about 8° C. The reaction mixture was stirred whilecooling with an ice-water bath for 30 minutes (NMR of a worked-upaliquot showed complete consumption of starting material). Added aqueous1 N Na₂S₂O₃ (330 mL), removed the cold bath and stirred for 20 minutes.The mixture was diluted with EtOAc and the layers were separated. Theorganic phase was washed with saturated aqueous NaHCO₃ (2×), water,brine, dried over MgSO₄, filtered through a short plug of silica, elutedwith EtOAc, and concentrated under reduced pressure to give (5) as avery dark amber oil (82.25 g, 77-94% HPLC purity). Carried forwardwithout further purification. LCMS (C18 column eluting with 10-90%CH₃CN/water gradient over 5 minutes with formic acid modifier) M+1:242.10 (2.89 min). ¹H NMR (300 MHz, CDCl₃) δ 7.22 (d, J=2.3 Hz, 1H),7.16 (dd, J =8.4, 2.3 Hz, 1H), 6.54 (d, J=8.4 Hz, 1H), 4.79-4.73 (m,1H), 4.15 (s, 2H), 4.10-4.01 (m, 1H), 3.93-3.85 (m, 1H), 2.26-2.13 (m,1H), 2.12-1.97 (m, 3H) ppm.

EXAMPLE 4 Preparation ofN-(4-bromo-2-nitro-6-tetrahydrofuran-2-yl-phenyl)-2,2,2-trifluoro-acetamide(6)

To trifluoroacetic anhydride (455.3 mL, 3.275 mol, Sigma-Aldrich 106232)stirring at 2° C. was slowly added4-bromo-2-tetrahydrofuran-2-yl-aniline (5) (79.29 g, 327.5 mmol) as athick oil via addition funnel over 15 minutes (reaction temperature roseto 14° C.). The remaining oil was rinsed into the reaction mixture withanhydrous 2-methyltetrahydrofuran (39.6 mL, Sigma-Aldrich 414247). Thecold bath was removed and ammonium nitrate (34.08 g, 425.8 mmol, Aldrich467758) was added. The reaction temperature rose to 40° C. over about 30minutes at which time a cold water bath was used to control the exothermand bring the reaction to room temperature. The cold bath was thenremoved and stirring continued for another 40 minutes (HPLC showed verylittle remaining un-nitrated material). The reaction mixture was slowlypoured into a stirring mixture of crushed ice (800 g). The solidprecipitate was collected by filtration, washed with water, saturatedaqueous NaHCO₃ (to pH 8), water again, and hexane. The wet solid wasdried first in a convection oven at 50° C. for several hours and thenunder reduced pressure in an oven at 40° C. overnight giving (6) as alight brown solid (77.86 g, 62% yield; 98% HPLC purity). LCMS (C18column eluting with 10-90% CH₃CN/water gradient over 5 minutes withformic acid modifier) M+1: 383.19 (3.27 min). ¹H NMR (300 MHz, CDCl₃) δ9.81 (s, 1H), 8.08 (d, J=2.2 Hz, 1H), 7.73 (d, J=2.2 Hz, 1H), 4.88 (dd,J=9.0, 6.5 Hz, 1H), 4.17-4.08 (m, 1H), 4.03-3.95 (m, 1H), 2.45-2.34 (m,1H), 2.17-2.06 (m, 2H), 1.96-1.83 (m, 1H) ppm.

EXAMPLE 5 Preparation of 4-bromo-2-nitro-6-tetrahydrofuran-2-yl-aniline(6a)

DissolvedN-(4-bromo-2-nitro-6-tetrahydrofuran-2-yl-phenyl)-2,2,2-trifluoro-acetamide(6) (54.00 g, 140.9 mmol) in 1,4-dioxane (162 mL) and added aqueous 6 MNaOH (70.45 mL, 422.7 mmol, JT-Baker 567202). The reaction mixture wasstirred at reflux for 2 days (HPLC showed complete conversion), allowedto cool, diluted with MTBE (800 mL), washed with water (2×200 mL),saturated aqueous NH₄Cl, water and brine. The mixture was dried overMgSO₄, filtered, and concentrated under reduced pressure to give (6a) asa dark amber oil (40.96 g, 93% yield; overall 92% HPLC plus NMR purity).LCMS (C18 column eluting with 10-90% MeOH/water gradient from 3-5minutes with formic acid modifier) M+1: 287.28 (3.44 min). ¹H NMR (300MHz, CDCl₃) δ 8.24 (d, J=2.4 Hz, 1H), 7.41 (d, J=2.3 Hz, 1H), 6.91 (s,2H), 4.80 (t, J=7.2 Hz, 1H), 4.14-4.05 (m, 1H), 3.98-3.90 (m, 1H),2.36-2.19 (m, 1H), 2.15-2.01 (m, 3H) ppm.

EXAMPLE 6 Preparation of2-[5-(4-amino-3-nitro-5-tetrahydrofuran-2-yl-phenyl)pyrimidin-2-yl]propan-2-ol(8)

Mixed 4-bromo-2-nitro-6-tetrahydrofuran-2-yl-aniline (6a) (40.40 g, 92%,129.5 mmol), 1,4-dioxane (260 mL, Sigma-Aldrich 360481),2-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl]propan-2-ol(7) (41.05 g, 155.4 mmol), and aqueous 2.7 M Na₂CO₃ (143.9 mL, 388.5mmol). A stream of nitrogen was bubbled through the stirring mixture for1 hr, followed by addition of tetrakis(triphenylphosphine)palladium (0)(7.48 g, 6.47 mmol, Strem 462150). The reaction mixture was stirred atreflux for 2 hrs (HPLC showed complete reaction), allowed to cool,diluted with EtOAc, washed with water, saturated aqueous NH₄Cl, brine,dried over MgSO₄, and filtered through a short plug of Florisil® elutingwith EtOAc. The filtrate was concentrated under reduced pressure givingadark brown oil. Dissolved in CH₂Cl₂ and eluted through a short plug ofsilica gel with CH₂Cl₂ and then EtOAc. The desired fraction wasconcentrated on a rotary evaporator until a precipitate formed giving athick brown slurry, which was triturated with MTBE. The solid wascollected by filtration, washed with MTBE, and dried under high vacuumgiving (8) as a yellow solid (35.14 g, 99+% HPLC purity). LCMS (C18column eluting with 10-90% CH₃CN/water gradient over 5 minutes withformic acid modifier) M+1: 345.00 (2.69 min). ¹H NMR (300 MHz, CDCl₃) δ8.88 (s, 2H), 8.36 (d, J=2.2 Hz, 1H), 7.56 (d, J=2.1 Hz, 1H), 7.09 (s,2H), 4.92 (t, J=7.2 Hz, 1H), 4.62 (s, 1H), 4.20-4.11 (m, 1H), 4.03-3.94(m, 1H), 2.39-2.26 (m, 1H), 2.23-2.08 (m, 3H), 1.64 (s, 6H) ppm. Thefiltrate was further concentrated and purified by ISCO silica gelchromatography eluting with 0 to 80% EtOAc/hexane giving a second cropof product (8) as an amber solid (4.46 g, 88% overall yield; 88% HPLCpurity.

EXAMPLE 7 Alternate preparation of2-[5-(4-amino-3-nitro-5-tetrahydrofuran-2-yl-phenyl)pyrimidin-2-yl]propan-2-ol(8)

MixedN-(4-bromo-2-nitro-6-tetrahydrofuran-2-yl-phenyl)-2,2,2-trifluoro-acetamide(6) (19.00 g, 49.59 mmol),2-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl]propan-2-ol(7) (14.41 g, 54.55 mmol), aqueous 2.7 M sodium carbonate (73.48 mL,198.4 mmol), and 1,4-dioxane (190 mL, Sigma-Aldrich 360481). A stream ofnitrogen was bubbled through the stirring mixture for 40 minutes,followed by addition of 1,1′-bis(diphenylphosphino)ferrocenedichloropalladium dichloromethane adduct (2.025 g, 2.480 mmol, Strem460450). The reaction mixture was stirred at reflux under N₂ for 7 hrs,added another 50 mL of saturated aqueous sodium carbonate, and refluxedfor another 16 hrs. The mixture was allowed to cool, then diluted withEtOAc (500 mL) and water (200 mL). The layers were separated and theaqueous phase extracted with EtOAc (200 mL). The combined organic phasewas washed with water (500 mL), brine (500 mL), dried over Na₂SO₄,filtered through a Florisil® plug, and concentrated on a rotaryevaporator to give crude (8) as an orange oil. Crude product waspurified by ISCO silica gel chromatography eluting with 20-90%EtOAc/hexane to give (8) as an orange solid (15.00 g, 87% yield; 81-88%purity). LCMS (C18 column eluting with 10-90% CH₃CN/water gradient over5 minutes with formic acid modifier) M+1: 345.35 (2.68 min). ¹H NMR (300MHz, CDCl₃) δ 8.88 (s, 2H), 8.36 (d, J=2.2 Hz, 1H), 7.56 (d, J=2.1 Hz,1H), 7.09 (s, 2H), 4.92 (t, J=7.2 Hz, 1H), 4.62 (s, 1H), 4.20-4.11 (m,1H), 4.03-3.94 (m, 1H), 2.39-2.26 (m, 1H), 2.23-2.08 (m, 3H), 1.64 (s,6H) ppm.

EXAMPLE 8 Preparation of2-[5-(3,4-diamino-5-tetrahydrofuran-2-yl-phenyl)pyrimidin-2-yl]propan-2-ol(9)

To a suspension of2-[5-(4-amino-3-nitro-5-tetrahydrofuran-2-yl-phenyl)pyrimidin-2-yl]propan-2-ol(8) (30.10 g, 87.41 mmol) and THF (90 mL) in a Parr bottle undernitrogen was added a slurry of 5% palladium on carbon (3.01 g, 50% wet,0.707 mmol, Aldrich 330116) in MeOH (90 mL, JT-Baker 909333), followedby NEt₃ (24.37 mL, 174.8 mmol, Sigma-Aldrich 471283). Placed the vesselon a Parr shaker and saturated with H₂. Added 45 psi H₂ and shook untilconsumption was complete (HPLC showed complete conversion). The reactionmixture was purged with nitrogen, filtered through Celite™ and rinsedwith EtOAc. The filtrate was re-filtered through a 0.5 micron glassfiber filter paper sandwiched between two P5 papers, and concentratedunder reduced pressure giving (9) as a light brown foam (28.96 g, 98%yield; 93% NMR purity). LCMS (C18 column eluting with 10-90% CH₃CN/watergradient over 5 minutes with formic acid modifier) M+1: 315.32 (1.54min). ¹H NMR (300 MHz, CDCl₃) δ 8.83 (s, 2H), 6.92 (d, J=1.8 Hz, 1H),6.88 (d, J=1.8 Hz, 1H), 4.90 (dd, J=7.9, 6.2 Hz, 1H), 4.72 (s, 1H), 4.18(s, 2H), 4.17-4.08 (m, 1H), 3.99-3.89 (m, 1H), 3.46 (s, 2H), 2.34-2.19(m, 1H), 2.17-2.05 (m, 3H), 1.63 (s, 6H) ppm.

EXAMPLE 9 Preparation of1-ethyl-3-[5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-tetrahydrofuran-2-yl-1H-benzimidazol-2-yl]urea(11)

To a stirring solution of2-[5-(3,4-diamino-5-tetrahydrofuran-2-yl-phenyl)pyrimidin-2-yl]propan-2-ol(9) (32.10 g, 102.1 mmol) in 1,4-dioxane (160.5 mL, Sigma-Aldrich360481) was added pH 3.5 buffer (240.8 mL), prepared by dissolving NaOActrihydrate (34.5 g) in 1N aqueous H₂SO₄ (240 mL). Added1-ethyl-3-(N-(ethylcarbamoyl)-C-methylsulfanyl-carbonimidoyl)urea (10)(28.46 g, 122.5 mmol, CB Research and Development) and stirred at refluxovernight (HPLC showed 99% consumption of starting diamine). Thereaction mixture was cooled to room temperature and poured portion-wise(frothing) into a stirring solution of aqueous saturated NaHCO₃ (480 mL)and water (120 mL) giving pH 8-9. This was stirred for 30 minutes, thesolid was collected by filtration, washed copiously with water toneutral pH, and then more sparingly with EtOH. The solid was dried underreduced pressure giving (11) as an off-white solid (34.48 g, 82% yield;99.4% HPLC purity). LCMS (C18 column eluting with 10-90% CH₃CN/watergradient over 5 minutes with formic acid modifier) M+1: 411.41 (1.73min). ¹H NMR (300 MHz, MeOD) δ 9.02 (s, 2H), 7.62 (s, 1H), 7.37 (s, 1H),5.31 (s, 1H), 4.23 (dd, J=14.5, 7.3 Hz, 1H), 4.01 (dd, J=15.0, 7.1 Hz,1H), 3.38-3.28 (m, 2H), 2.58-2.46 (m, 1H), 2.16-2.05 (m, 2H), 2.02-1.88(m, 1H), 1.63 (s, 6H), 1.22 (t, J=7.2 Hz, 3H) ppm.

EXAMPLE 10 Chiral chromatographic isolation of1-ethyl-3-[5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(12)

A racemic sample of1-ethyl-3-[5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-tetrahydrofuran-2-yl-1H-benzimidazol-2-yl]urea(11) (24.60 g) was resolved on a CHIRALPAK® IC® column (by ChiralTechnologies) eluting with DCM/MeOH/TEA (60/40/0.1) at 35° C. giving thedesired enantiomer (12) as a white solid (11.35 g, 45% yield; 99+% HPLCpurity, 99+% ee). Analytical chiral HPLC retention time was 6.2 min(CHIRALPAK® IC® 4.6×250 mm column, 1 mL/min flow rate, 30° C.).

The structure and absolute stereochemistry of 12 were confirmed bysingle-crystal x-ray diffraction analysis. Single crystal diffractiondata were acquired on a Bruker Apex II diffractometer equipped withsealed tube Cu K-alpha source (Cu Kα radiation, γ=1.54178 Å) and an ApexII CCD detector. A crystal with dimensions of ½×0.05×0.05 mm wasselected, cleaned using mineral oil, mounted on a MicroMount andcentered on a Bruker APEXII system. Three batches of 40 frames separatedin reciprocal space were obtained to provide an orientation matrix andinitial cell parameters. Final cell parameters were obtained and refinedafter data collection was completed based on the full data set. Based onsystematic absences and intensities statistics the structure was solvedand refined in acentric P2₁ space group.

A diffraction data set of reciprocal space was obtained to a resolutionof 0.9 Å using 0.5° steps using 60 s exposure for each frame. Data werecollected at 100 (2) K. Integration of intensities and refinement ofcell parameters were accomplished using APEXII software. Observation ofthe crystal after data collection showed no signs of decomposition. Asshown in FIG. 1, there are two symmetry independent molecules in thestructure and both symmetry independent molecules are R isomers.

The data were collected, refined and reduced using the Apex II software.The structure was solved using the SHELXS97 (Sheldrick, 1990);program(s) and the structure refined using the SHELXL97 (Sheldrick,1997) program. The crystal shows monoclinic cell with P2₁ space group.The lattice parameters are a=9.8423(4) Å, b=10.8426(3) Å, c=19.4441 (7)Å, β=102.966(3)°. Volume=2022.09(12) Å³.

EXAMPLE 11 Preparation of the methanesulfonic acid salt of1-ethyl-3-[5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(13)

A stirring suspension of1-ethyl-3-[5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(12) (9.32 g, 22.71 mmol) in absolute ethanol (93.2 mL) was cooled withan ice-water bath. Added methanesulfonic acid (1.548 mL, 23.85 mmol,Sigma-Aldrich 471356), removed cold bath and stirred at room temperaturefor 20 minutes. It was concentrated on a rotary evaporator at 35° C. toa thick slurry, diluted with EtOAc, collected the solid by filtration,washed with EtOAc, and dried under reduced pressure giving an initialcrop of (13) as a white solid (8.10 g). The filtrate was concentrated ona rotavap giving a yellowish glassy foam, which was dissolved in EtOH,concentrated to a solid slurry, triturated with EtOAc/Et₂O, andcollected by filtration. The solid was washed with EtOAc/Et₂O, combinedwith the first crop, and dried under reduced pressure giving (13) as awhite solid (9.89 g, 86% yield; 99+% HPLC purity, 99+% ee).

Analytical chiral HPLC shows one enantiomer with retention time of 6.3min eluting with DCM/MeOH/TEA (60/40/0.1) on a CHIRALPAK® IC® 4.6×250 mmcolumn with 1 mL/min flow rate at 30° C. LCMS (C18 column eluting with10-90% CH₃CN/water gradient over 5 minutes with formic acid modifier)M+1: 411.53 (174 min). ¹H NMR (300 MHz, MeOD) δ 9.07 (s, 2H), 7.79 (s,1H), 7.62 (s, 1H), 5.30 (t, J=7.3 Hz, 1H), 4.24 (dd, J=14.6, 7.3 Hz,1H), 4.04 (dd, J=15.0, 7.6 Hz, 1H), 3.40-3.30 (m, 2H), 2.72 (s, 3H),2.65-2.54 (m, 1H), 2.20-2.07 (m, 2H), 2.04-1.90 (m, 1H), 1.64 (s, 6H),1.23 (t, J=7.2 Hz, 3H) ppm.

EXAMPLE 12 Preparation of 2-(2-fluoro-6-nitro-phenyl)-2,3-dihydrofuran(15A) and 2-(2-fluoro-6-nitro-phenyl)-2,5-dihydrofuran (15B)

2-Bromo-1-fluoro-3-nitro-benzene (14) (200.3 g, 98%, 892.3 mmol, BoscheF6657), 1,4-dioxane (981.5 mL, Sigma-Aldrich 360481), and2,3-dihydrofuran (2) (341.1 mL, 99%, 4.462 mol, Aldrich 200018) werecharged in a reaction flask, followed by N,N-diisopropylethylamine(155.4 mL, 892.3 mmol, Sigma-Aldrich 550043) andbromo(tri-tert-butylphosphine)palladium(I) dimer (6.936 g, 8.923 mmol,Johnson Matthey C4099). The mixture was stirred at reflux for 2 hrs(HPLC showed 98% consumption of starting arylbromide). It was allowed tocool, the precipitate was removed by filtration, rinsed with EtOAc, andthe filtrate concentrated in vacuo to a dark reddish brown semi-solidoil. This was dissolved in CH₂Cl₂, eluted through a plug of silica withCH₂Cl₂, and concentrated in vacuo giving a mixture of 15A and 15B as adark amber oil (291.3 g). The crude product was carried forward withoutfurther purification. The major product was2-(2-fluoro-6-nitrophenyl)-2,3-dihydrofuran (15A) (96%): LCMS (C18column eluting with 10-90% CH₃CN/ water gradient over 5 minutes withformic acid modifier) M+1: 210.23 (3.13 min); ¹H NMR (300 MHz, CDCl₃) δ7.54 (dt, J=8.0, 1.2 Hz, 1H), 7.43 (td, J=8.2, 5.2 Hz, 1H), 7.32 (ddd, J=9.7, 8.3, 1.3 Hz, 1H), 6.33 (dd, J=4.9, 2.4 Hz, 1H), 5.80 (t, J=10.9Hz, 1H), 5.06 (q, J=2.4 Hz, 1H), 3.18-3.07 (m, 1H), 2.94-2.82 (m, 1H)ppm. The minor product was 2-(2-fluoro-6-nitro-phenyl)-2,5-dihydrofuran(15B) (4%): GCMS (Agilent HP-5MS 30 m×250×μm×0.25 μm column heating at60° C. for 2 min to 300° C. over 15 min with a 1 mL/min flow rate) M+1:210 (11.95 min). ¹H NMR (300 MHz, CDCl₃) δ 7.47 (d, J=8.0 Hz, 1H),7.43-7.34 (m, 1H), 7.30-7.23 (m, 1H), 6.21-6.15 (m, 1H), 6.11-6.06 (m,1H), 5.97-5.91 (m, 1H), 4.89-4.73 (m, 2H) ppm.

EXAMPLE 13 Preparation of 3-fluoro-2-tetrahydrofuran-2-yl-aniline (16)

Placed 5% palladium on carbon (37.3 g, 50% wet, 8.76 mmol, Aldrich330116) in a Parr bottle under nitrogen, followed by MeOH (70 mL,JT-Baker 909333). Added the crude mixture of2-(2-fluoro-6-nitro-phenyl)-2,3-dihydrofuran and2-(2-fluoro-6-nitrophenyl)-2,5-dihydrofuran (15A&15B) (186.6 g, 892.1mmol) dissolved in MeOH (117 mL), followed by NEt₃ (124.3 mL, 892.1mmol, Sigma-Aldrich 471283). Placed the vessel on a Parr shaker andsaturated with H₂. After adding 45 psi H₂, the reaction mixture wasshaken until consumption of the starting material was complete (HPLC andLCMS showed complete reaction). The reaction mixture was purged withnitrogen, filtered through Celite™ and rinsed with EtOAc. The filtratewas concentrated on a rotary evaporator giving a brown oil, which wasdissolved in Et₂O and washed with water (2×). The ether phase wasextracted with aqueous 1 N HCl (5×250 mL), which was washed with Et₂O(3×) and then basified with aqueous 6 N NaOH to pH 12-14. The basicaqueous phase was extracted with CH₂Cl₂(4×), and the combined organicextract washed with saturated aqueous NH₄Cl, dried over MgSO₄, andfiltered through a pad of silica eluting with CH₂Cl₂ to 25%EtOAc/hexane. The desired filtrate was concentrated under reducedpressure giving 16 as a light brown oil (121.8 g, 84% GCMS plus NMRpurity). GCMS (Agilent HP-5MS 30 m×250 μm×0.25 μm column heating at 60°C. for 2 min to 300° C. over 15 min with a 1 mL/min flow rate) M+1:182.0 (11.44 min). LCMS (C18 column eluting with 10-90% CH₃CN/watergradient over 5 minutes with formic acid modifier) M+1: 182.10 (2.61min). ¹H-NMR (300 MHz, CDCl₃) δ 6.97 (td, J=8.1, 6.3 Hz, 1H), 6.43-6.35(m, 2H), 5.21-5.13 (m, 1H), 4.54 (s, 2H), 4.16-4.07 (m, 1H), 3.90-3.81(m, 1H), 2.23-2.00 (m, 4H) ppm. Additional crops were obtained asfollows: the combined ether phase was washed with saturated aqueousNaHCO₃, brine, dried over Na₂SO₄, decanted, and concentrated underreduced pressure. The oil was vacuum distilled (ca. 15 torr) collectingthe distillate at 101-108° C. To a stirring solution of the distilledoil in EtOH (1 volume) at 2° C. was slowly added 5 M HCl (1 eq) iniPrOH. The resulting suspension was brought to room temperature, dilutedwith EtOAc (3 volumes, vol/vol), and stirred for 2 hrs. The white solidwas collected by filtration, washed with EtOAc, and dried under reducedpressure giving a second crop of product as the HCl salt. The motherliquor was concentrated to a slurry, diluted with EtOAc and the solidcollected by filtration, washed with EtOAc, and dried in vacuo givingthe HCl salt as a third crop of the product. LCMS (C18 column elutingwith 10-90% CH₃CN/water gradient over 5 minutes with formic acidmodifier) M+1: 182.10 (2.58 min). ¹H NMR (300 MHz, CDCl₃) δ 10.73 (br.s,3H), 7.66 (d, J=8.1 Hz, 1H), 7.33 (td, J=8.2, 5.9 Hz, 1H), 7.13-7.05 (m,1H), 5.26 (dd, J=9.0, 6.5 Hz, 1H), 4.38-4.28 (m, 1H), 4.00-3.91 (m, 1H),2.59-2.46 (m, 1H), 2.30-1.95 (m, 3H) ppm. The overall yield from thethree crops was 76%.

EXAMPLE 14 Preparation of4-bromo-3-fluoro-2-tetrahydrofuran-2-yl-aniline (17)

To a stirring solution of 3-fluoro-2-tetrahydrofuran-2-yl-aniline (16)(131.9 g, 92%, 669.7 mmol) in methyl tert-butyl ether (1.456 L) andacetonitrile (485 mL) cooled to −20° C. was added N-bromosuccinimide(120.4 g, 99%, 669.7 mmol, Aldrich B81255) in 3 portions maintaining areaction temperature below about −15° C. After complete additionstirring was continued at −15 to −10° C. for 30 minutes. ¹H NMR of aworked-up aliquot showed 96% consumption of starting aniline so addedanother 4.82 g NBS and stirred at −10° C. for another 30 minutes.Aqueous 1 N Na₂S₂O₃ (670 mL) was added to the reaction mixture. The coldbath was removed, the mixture stirred for 20 minutes, then diluted withEtOAc. The layers were separated and the organic phase was washed withsaturated aqueous NaHCO₃ (2×), water, brine, dried over Na₂SO₄,decanted, and concentrated under reduced pressure giving a dark amberoil. The residue was diluted with hexane and eluted through a short plugof silica eluting with 25% EtOAc/hexane to 50% EtOAc/hexane. The desiredfiltrate was concentrated in vacuo giving 17 as a dark amber oil (182.9g, 90% yield; 86% NMR purity). LCMS (C18 column eluting with 10-90%CH₃CN/water gradient over 5 minutes with formic acid modifier) M+1:260.12 (3.20 min). ¹H NMR (300 MHz, CDCl₃) δ 7.15 (dd, J=8.6, 7.6 Hz,1H), 6.30 (dd, J=8.7, 1.3 Hz, 1H), 5.19-5.12 (m, 1H), 4.58 (s, 2H),4.16-4.07 (m, 1H), 3.90-3.81 (m, 1H), 2.23-1.99 (m, 4H) ppm.

EXAMPLE 15 Preparation ofN-(4-bromo-3-fluoro-6-nitro-2-tetrahydrofuran-2-yl-phenyl)-2,2,2-trifluoro-acetamide(18)

To trifluoroacetic anhydride (565.3 mL, 4.067 mol, Sigma-Aldrich 106232)stirring at 2° C. was slowly added neat4-bromo-3-fluoro-2-tetrahydrofuran-2-yl-aniline (17) (123.0 g, 86%,406.7 mmol) as a thick oil via addition funnel over about 20 minutes(reaction temperature rose to 13° C.). The remaining oil was rinsed intothe reaction mixture with anhydrous THF (35 mL). The cold bath wasremoved and the reaction was heated to 35° C., followed by portion-wiseaddition of NH₄NO₃ (4.88 g×20 portions, 1.22 mol, Sigma-Aldrich A7455)over 2.5 hrs maintaining the reaction temperature between 30 and 41° C.using an ice-water bath only as needed to control the exotherm. Aftercomplete addition the reaction mixture was stirred for another 10minutes (HPLC showed reaction 99% complete). It was slowly poured intocrushed ice (1.23 kg) and stirred for 1 hr to allow formation of afilterable solid precipitate, which was collected and washed with water,sparingly with saturated aqueous NaHCO₃, and water again (to pH 7). Theproduct was dried in a convection oven overnight at 40° C. and thenunder reduced pressure in an oven at 50° C. overnight giving 18 as abeige solid (152.5 g, 90% yield; 96% HPLC purity). LCMS (C18 columneluting with 10-90% CH₃CN/water gradient over 5 minutes with formic acidmodifier) M+1: 401.30 (3.41 min). ¹H NMR (300 MHz, CDCl₃) δ 10.56 (s,1H), 8.19 (d, J=6.6 Hz, 1H), 5.22 (dd, J=10.3, 6.4 Hz, 1H), 4.22 (dd,J=15.8, 7.2 Hz, 1H), 3.99 (dd, J=16A, 7.5 Hz, 1H), 2.50-2.38 (m, 1H),2.22-2.11 (m, 2H), 1.86-1.71 (m, 1H) ppm.

EXAMPLE 16 Preparation of4-bromo-3-fluoro-6-nitro-2-tetrahydrofuran-2-yl-aniline (19)

A reaction flask was charged withN-(4-bromo-3-fluoro-6-nitro-2-tetrahydrofuran-2-yl-phenyl)-2,2,2-trifluoro-acetamide(18) (242.3 g, 604.1 mmol), 1,4-dioxane (1.212 L), aqueous 2 M sulfuricacid (362.4 mL, 724.9 mmol), and stirred at reflux for 5 days (HPLCshowed 98% conversion). Allowed to cool, diluted with EtOAc, neutralizedwith saturated aqueous NaHCO₃, separated the layers, and re-extractedthe aqueous phase with EtOAc (2×). The combined organic phase was washedwith brine (2×), dried over MgSO₄, filtered and concentrated in vacuogiving 19 as a greenish brown solid (181.7 g, 94% yield; 95% HPLCpurity). The product was carried to the next step without furtherpurification. LCMS (C18 column eluting with 10-90% CH₃CN/water gradientover 5 minutes with formic acid modifier) M+1: 305.20 (3.63 min). ¹H NMR(300 MHz, CDCl₃) δ 8.35 (d, J=7.3 Hz, 1H), 7.45 (s, 2H), 5.23-5.16 (m,1H), 4.23-4.14 (m, 1H), 3.93-3.84 (m, 1H), 2.31-1.96 (m, 4H) ppm.

EXAMPLE 17 Preparation of2-[5-(4-amino-2-fluoro-5-nitro-3-tetrahydrofuran-2-yl-phenyl)pyrimidin-2-yl]propan-2-ol(20)

To a stirring solution of4-bromo-3-fluoro-6-nitro-2-tetrahydrofuran-2-yl-aniline (19) (525.0 g,1.721 mol, Bridge Organics Co.) in 1,4-dioxane (4.20 L, Sigma-Aldrich360481) was added a 1.2 M aqueous solution of NaHCO₃ (4.302 L, 5.163mol). A stream of nitrogen was bubbled through the stirring mixture for2 hrs, followed by addition of2-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl]propan-2-ol(7) (545.4 g, 2.065 mol, Bridge Organics Co.) and1,1′-bis(diphenylphosphino)ferrocene dichloropalladium dichloromethaneadduct (42.16 g, 51.63 mmol, Strem 460450). The reaction mixture wasstirred at reflux overnight, allowed to cool, diluted with EtOAc (8.4L), and the layers were separated. The organic phase was washed withsaturated aqueous NH₄Cl and then brine. The aqueous phase wasre-extracted with EtOAc (4 L) and washed this organic extract withbrine. The combined organic phase was dried over MgSO₄, filtered througha short plug of Florisil®, eluted with EtOAc, and the filtrateconcentrated on a rotary evaporator giving a dark brown wet solid. Thiswas dissolved in CH₂Cl₂, loaded on a pad of silica gel, eluted withhexane, then 25% EtOAc/hexane, and then 50% EtOAc/hexane. The desiredfiltrate was concentrated on a rotary evaporator to a thick suspension,and the solid was collected by filtration, triturated with MTBE, anddried in vacuo giving 20 as a bright yellow solid (55.8% yield, 90-97%HPLC purity). The filtrate was concentrated and the above purificationwas repeated giving a second crop of 20 as a bright yellow solid (19.7%yield). The filtrate was again concentrated giving a dark brown oil andthis was loaded on a silica column with toluene and minimal CH₂Cl₂. Itwas eluted with EtOAc/hexane (0% to 50%). The desired fractions wereconcentrated to a slurry and diluted with MTBE/hexane. The solid wascollected by filtration and washed with minimal MTBE giving a third cropof 20 as a bright yellow solid (4.9% yield) with an overall yield of 80%from the three crops. LCMS (C18 column eluting with 10-90% CH₃CN/watergradient over 5 minutes with formic acid modifier) M+1: 363.48 (2.95min). ¹H NMR (300 MHz, CDCl₃) δ 8.84 (d, J=1.6 Hz, 2H), 8.27 (d, J=8.0Hz, 1H), 7.62 (s, 2H), 5.31-5.24 (m, 1H), 4.63 (s, 1H), 4.27-4.18 (m,1H), 3.97-3.87 (m, 1H), 2.33-2.05 (m, 4H), 1.64 (s, 6H) ppm.

EXAMPLE 18 Preparation of2-[5-(4,5-diamino-2-fluoro-3-tetrahydrofuran-2-yl-phenyl)pyrimidin-2-yl]propan-2-ol(21)

Placed 5% palladium on carbon (14.21 g, 50% wet, 3.339 mmol, Aldrich330116) in a Parr bottle under nitrogen, followed by MeOH (242 mL,JT-Baker 909333) and NEt₃ (46.54 mL, 333.9 mmol, Sigma-Aldrich 471283).Dissolved2-[5-(4-amino-2-fluoro-5-nitro-3-tetrahydrofuran-2-yl-phenyl)pyrimidin-2-yl]propan-2-ol(20) (121.0 g, 333.9 mmol) in hot THF (360 mL), allowed to cool, addedto the reaction mixture, and rinsed with another portion of THF (124mL). Placed the vessel on a Parr shaker and saturated with H₂. Added 45psi H₂ and shook until consumption was complete (HPLC and LCMS showedcomplete reaction). The reaction mixture was purged with nitrogen,filtered through Celite™ and rinsed with EtOAc. It was re-filteredthrough paper (glass microfibre) and the filtrate concentrated in vacuo.Repeated the reaction three more times on the same scale and the batcheswere combined giving 21 as a brown solid (447 g, 99% yield; 93% HPLCpurity). LCMS (C18 column eluting with 10-90% CH₃CN/water gradient over5 minutes with formic acid modifier) M+1: 333.46 (1.79 min). ¹H NMR (300MHz, CDCl₃) δ 8.81 (d, J=1.4 Hz, 2H), 6.69 (d, J=7.3 Hz, 1H), 5.27-5.20(m, 1H), 4.73 (s, 1H), 4.70 (s, 2H), 4.23-4.14 (m, 1H), 3.94-3.86 (m,1H), 3.22 (s, 2H), 2.32-2.22 (m, 1H), 2.18-199 (m, 3H), 1.63 (s, 6H)ppm.

EXAMPLE 19 Preparation of1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-tetrahydrofuran-2-yl-1H-benzimidazol-2-yl]urea(22)

To a stirring suspension of2-[5-(4,5-diamino-2-fluoro-3-tetrahydrofuran-2-yl-phenyl)pyrimidin-2-yl]propan-2-ol(21) (111.3 g, 334.9 mmol) and 1,4-dioxane (556.5 mL, Sigma-Aldrich360481) was added1-ethyl-3-(N-(ethylcarbamoyl)-C-methylsulfanyl-carbonimidoyl)urea (10)(93.36 g, 401.9 mmol, CB Research and Development) followed by a pH 3.5buffer (1.113 L), prepared by dissolving NaOAc trihydrate (158.1 g) in1N aqueous H₂SO₄ (1.100 L). The reaction mixture was stirred at refluxovernight (HPLC showed complete conversion), cooled to room temperature,and poured portion-wise (frothing) into a stirring solution of aqueoussaturated NaHCO₃ (2.23 L) giving pH 8-9. This was stirred for 30minutes, the solid was collected by filtration, washed copiously withwater to neutral pH, and then more sparingly with EtOH. The solid wasdried under reduced pressure giving 22 as an off-white yellowish solid(135.2 g, 94% yield; 99% HPLC purity). LCMS (C18 column eluting with10-90% CH₃CN/water gradient over 5 minutes with formic acid modifier)M+1: 429.58 (2.03 min). ¹H NMR (300 MHz, MeOD) δ 8.95 (d, J=1.6 Hz, 2H),7.45 (d, J=6.5 Hz, 1H), 5.38 (br.s, 1H), 4.27 (dd, J=14.9, 7.1 Hz, 1H),4.01 (dd, J=15.1, 7.0 Hz, 1H), 3.37-3.29 (m, 2H), 2.55 (br.s, 1H),2.19-2.07 (m, 2H), 2.02-1.82 (br.s, 1H), 1.63 (s, 6H), 1.21 (t, J=7.2Hz, 3H) ppm.

EXAMPLE 20 Chiral chromatographic isolation of1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(23)

A racemic sample of1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-tetrahydrofuran-2-yl-1H-benzimidazol-2-yl]urea(22) (133.60 g) was resolved on a CHIRALPAK® IC® column (by ChiralTechnologies) eluting with DCM/MeOH/TEA (60/40/0.1) at 25° C. giving thedesired enantiomer 23 as an off-white solid (66.8 g, 45% yield; 99.8%HPLC purity, 99+% ee). Analytical chiral HPLC retention time was 7.7 min(CHIRALPAK® IC® 4.6×250 mm column, 1 mL/min flow rate, 30° C.). Thesolid was suspended in 2:1 EtOH/Et₂O (5 volumes), stirred for 10minutes, collected by filtration, washed with 2:1 EtOH/Et₂O, and driedunder reduced pressure giving a white solid (60.6 g).

The structure and absolute stereochemistry of 23 were confirmed bysingle-crystal x-ray diffraction analysis. Single crystal diffractiondata were acquired on a Bruker Apex II diffractometer equipped withsealed tube Cu K-alpha source (Cu Kα radiation, γ=1.54178 Å) and an ApexII CCD detector. A crystal with dimensions of 0.15×0.15×0.10 mm wasselected, cleaned using mineral oil, mounted on a MicroMount andcentered on a Bruker APEXII system. Three batches of 40 frames separatedin reciprocal space were obtained to provide an orientation matrix andinitial cell parameters. Final cell parameters were obtained and refinedafter data collection was completed based on the full data set. Based onsystematic absences and intensities statistics the structure was solvedand refined in acentric P2₁ space group.

A diffraction data set of reciprocal space was obtained to a resolutionof 0.85 Å using 0.5° steps using 30 s exposures for each frame. Datawere collected at 100 (2) K. Integration of intensities and refinementof cell parameters were accomplished using APEXII software. Observationof the crystal after data collection showed no signs of decomposition.As shown in FIG. 2, there are two symmetry independent molecules in thestructure and both symmetry independent molecules are R isomers.

The data were collected, refined and reduced using the Apex II software.The structure was solved using the SHELXS97 (Sheldrick, 1990);program(s) and the structure refined using the SHELXL97 (Sheldrick,1997) program. The crystal shows monoclinic cell with P2₁ space group.The lattice parameters are a=9.9016(2) Å, b=10.9184(2) Å, c=19.2975(4)Å, β=102.826(1)°. Volume=2034.19(7) Å³.

EXAMPLE 21 Preparation of the methanesulfonic acid salt of1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(23A)

To a stirring suspension of1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(23) (15.05 g, 35.13 mmol) in dichloromethane (60 mL, J. T. Baker931533) and absolute ethanol (15 mL, Pharmco-AAPER 111000200) was addedmethanesulfonic acid (2.392 mL, 36.89 mmol, Sigma-Aldrich 471356).Stirred at room temperature until a clear solution was observed. Addedheptane (300 mL) slowly over about 1 hr and collected the solidprecipitate by filtration (using a Whatman qualitative #3 paper on topof a Whatman GF/F glass microfibre paper). Dried under reduced pressurein a vacuum oven (desiccated with calcium sulfate and potassiumhydroxide) overnight at 40° C. giving 23A as a white solid (13.46 g,99+% HPLC purity, 99+% ee). Analytical chiral HPLC shows one enantiomerwith retention time of 8.6 min eluting with CH₂Cl₂/MeOH/TEA (60/40/0.1)on a CHIRAL PAK® IC® 4.6×250 mm column with 1 mL/min flow rate at 30° C.A second crop of white solid product 23A (4.36 g, 98% HPLC purity, 99+%ee) was obtained from the filtrate. LCMS (C18 column eluting with 10-90%CH₃CN/water gradient over 5 minutes with formic acid modifier) M+1:429.58 (2.03 min). ¹H NMR (300 MHz, MeOD) δ 9.00 (d, J=1.6 Hz, 2H), 7.67(d, J=6.1 Hz, 1H), 5.39 (t, J=7.7 Hz, 1H), 4.30 (dd, J=14.9, 6.9 Hz,1H), 4.03 (dd, J=14.8, 7.7 Hz, 1H), 3.40-3.31 (m, 2H), 2.72 (s, 3H),2.70-2.60 (m, 1H), 2.21-2.08 (m, 2H), 1.98-1.84 (m, 1H), 165 (s, 6H),122 (t, J=7.2 Hz, 3H) ppm.

The(R)-1-ethyl-3-(6-fluoro-5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea23 may then be converted to the phosphate or phosphate salt prodrugsaccording to the schemes set forth below.

Compounds of formula (IB) may be prepared from compound 23 as shown inScheme 1. In Step 1, compound 23 is treated with dibenzylN,N-diisopropylphosphoramidite and tetrazole, followed bymeta-chloroperoxybenzoic acid (mCPBA), to afford dibenzyl phosphate 24.In Step 2, hydrogenolysis of 24 in the presence of M⁺ OH⁻ or D²⁺(OH⁻)₂affords the dianionic form of the compound of formula (IB)(X═—PO(O⁻)₂.2M⁺ or —PO(O⁻)₂.D²⁺). The free acid form of the compound offormula (IB) (X═PO(OH)₂) may be obtained by treating the dianionic formwith aqueous acid. The monoanionic form of the compound of formula (IB)(X═PO(OH)O⁻M⁺) may be obtained by treating the free acid form with oneequivalent of M⁺OH⁻.

Alternatively, the compounds of formula (IB) may be prepared fromcompound 23 as shown in Scheme 2. In Step 1, compound 23 is treated withdi-tert-butyl dicarbonate (Boc₂O) to afford protected benzimidazolecompound 25. In Step 2, compound 25 is treated with dibenzylN,N-diisopropylphosphoramidite and tetrazole, followed by mCPBA, toafford protected dibenzyl phosphate 26. In Step 3, compound 26 istreated with trifluoroacetic acid (TFA) to remove the protecting groupand afford dibenzyl phosphate 24. In Step 4, hydrogenolysis of 24 in thepresence of M⁺OH⁻ or D²⁺(OH⁻)₂ affords the dianionic form of thecompound of formula (IB) (X═—PO(O⁻)₂.2M⁺ or —PO(O⁻)₂.D²⁺). The free acidform of the compound of formula (IB) (X═PO(OH)₂) may be obtained bytreating the dianionic form with aqueous acid. The monoanionic form ofthe compound of formula (I) (X═PO(OH)O⁻M⁺) may be obtained by treatingthe free acid form with one equivalent of M⁺OH⁻.

The compounds of formula (IB) may also be prepared from compound 23 asshown in Scheme 3. In Step 1, compound 23 is treated with twoequivalents of Boc₂O in the presence of N,N-dimethylaminopyridine (DMAP)to afford bis-protected benzimidazole compound 28. In Step 2, compound28 is treated with dibenzyl N,N-diisopropylphosphoramidite andtetrazole, followed by mCPBA, to afford bis-protected dibenzyl phosphate29. In Step 3, compound 29 is treated with TFA to remove the protectinggroups. Treatment of the resulting intermediate with aqueous M⁺ OH⁻ orD²⁺(OH⁻)₂ affords the dianionic form of the compound of formula (IB)(X═—PO(O⁻)₂.2M⁺ or —PO(O⁻)₂.D²⁺). The free acid form of the compound offormula (IB) (X═PO(OH)₂) may be obtained by treating the dianionic formwith aqueous acid. The monoanionic form of the compound of formula (I)(X═PO(OH)O⁻M⁺) may be obtained by treating the free acid form with oneequivalent of M⁺OH⁻.

EXAMPLE 22 Preparation of (R)-dibenzyl2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-ylphosphate (24)

To1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(23) (10.24 g, 23.66 mmol) in a 1 L round bottom flask under N₂ at 23°C. was added DMF (200 mL) followed by a solution of tetrazole (105.2 mLof 0.45 M in MeCN, 47.32 mmol) followed byN-dibenzyloxyphosphanyl-N-isopropyl-propan-2-amine (12.26 g, 11.93 mL,35.49 mmol). After 4.5 h moreN-dibenzyloxyphosphanyl-N-isopropyl-propan-2-amine (4 mL) was added.After stirring a further 16 h the reaction was cooled to 0° C.(ice-water bath) then treated with mCPBA (15.17 g, 61.52 mmol). Themixture was stirred at 0° C. for 30 min then at 23° C. for 30 min afterwhich the reaction mixture was partitioned between water (400 mL) andEtOAc (700 mL). The organic layer was separated, washed with saturatedaqueous sodium bicarbonate (500 mL), 10% aqueous sodium bisulfite (500mL), saturated aqueous sodium bicarbonate (500 mL), and brine (500 mL)then dried (magnesium sulfate), filtered and concentrated. The residuewas purified by MPLC using an ISCO COMBIFLASH brand flash chromatographypurification system (330 g column) eluting with a 0-10% EtOH in DCMlinear gradient over 16.5 column volumes at a 200 mL/min flow rate.After concentration in vacuo, (R)-dibenzyl2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-ylphosphate(24) (13.89 g, 20.17 mmol, 85.27%) was obtained as a whitesolid. ESMS (M+1)=689.5; ¹H NMR (300 MHz, CD₃OD) δ 8.88 (d, J=1.6 Hz,2H), 7.37 (d, J=6 Hz, 1H), 7.30 (m, 10H), 5.38-5.33 (m, 1H), 5.12-5.01(m, 4H), 4.24 (dd, J=6.8, 14.9 Hz, 1H), 3.98 (dd, J=6.9, 15.1 Hz, 1H),3.35-3.27 (m, 3H), 2.52 (q, J=5.9 Hz, 1H), 2.14-2.05 (m, 2H), 191 (s,6H) and 1.22-1.14 (m, 3H) ppm.

EXAMPLE 23 Preparation of disodium(R)-2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-ylphosphate (W)

A 1 L Parr vessel was charged with water (200 mL), Pd/C (4 g, 10 wt %dry basis, wet, Degussa type), (R)-dibenzyl2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-ylphosphate(24) (13.89 g, 20.17 mmol), EtOH (400 mL) and aqueous 1 M NaOH(40.34 mL, 40.34 mmol). The resulting mixture was hydrogenated under 50psi H₂ on a Parr shaker apparatus for 40 min. The reaction mixture wasfiltered through a 0.22 μm polyethersulfone (PES) membrane giving a darkcolored filtrate. A water rinse resulted in more dark material crossingthe filter membrane. The resulting filtrate was passed through a pad ofCelite and the dark material did not elute until the pad was rinsed withwater. The resulting dark solution (approx. 700 mL) was diluted withthree volumes of EtOH (2100 mL), filtered through a 0.22 μm PES membrane(using 4 disposable Corning polystyrene filter systems, #431098) and thefiltrate concentrated in vacuo. The resulting residue was dissolved inwater (100 mL) and EtOH (300 mL), filtered through a 0.22 μm PESmembrane to give a clear yellow solution, then passed through a Celiteplug (26 mm diameter×60 mm height, pre-wet with EtOH) rinsing with EtOH(50 mL) and the filtrate then concentrated. The resulting residue wasdissolved in water (250 mL) in a 1 L round bottom flask, then 1 Maqueous HCl (40 mL) was slowly added over 15 min with stirring to give aslurry of white solid. Twenty minutes following completion of the HCladdition, the solid was collected via filtration through a 0.22 μm PESmembrane. The collected solid was washed with water (100 mL), thentransferred (still wet) to a 1 L round bottom flask and slurried in MeOH(150 mL) for 30 min. The resulting fine white precipitate was collectedvia filtration then dried in vacuo overnight. The resulting free acid(9.17 g, 18.0 mmol) was treated with water (80 mL) then 1.0 N aq NaOH(36.0 mL, 2 equiv). The resulting solution was frozen and lyophilized togivedisodium[1-[5-[2-(ethylcarbamoylamino)-6-fluoro-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-5-yl]pyrimidin-2-yl]-1-methyl-ethyl]phosphate(W) (10.206 g, 18.08 mmol, 89.66%) as a pale, cream-colored solid withconsistent analytical data. ESMS (M+1)=509.4; ¹H NMR (300 MHz, D₂O) δ8.58 (s, 2H), 6.92 (d, J=6.3 Hz, 1H), 5.13 (t, J=7.5 Hz, 1H), 3.98-3.81(m, 2H), 3.04 (q, J=7.2 Hz, 2H), 2.26 (t, J=5.7 Hz, 1H), 1.97-1.92 (m,2H), 1.67 (s, 6H) and 1.01 (t, J=7.2 Hz, 3H) ppm.

EXAMPLE 24 Preparation ofBoc-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(25)

To a solution/suspension of1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(23) (10.72 g, 25.02 mmol) in DMF (250 mL) at 23° C. was added Boc₂O(6.11 g, 28.00 mmol). After 2 hours, 2 M ammonia in MeOH (2 mL) wasadded to quench any excess Boc₂O. The quenched reaction mixture waspartitioned between EtOAc and water (400 mL each), the organic layerseparated, washed with water (2×400 mL) and brine (400 mL), then driedover MgSO₄, filtered and concentrated to giveBoc-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(25) (12.69 g, 23.58 mmol, 94.26%) which was used without furtherpurification. ESMS (M+1)=529.3; ¹H NMR (300.0 MHz, CDCl₃) δ 9.50 (s,1H), 9.02 (t, J=5.3 Hz, 1H), 8.91 (d, J=1.6 Hz, 2H), 7.74 (d, J=6.5 Hz,1H), 5.58 (t, J=7.8 Hz, 1H), 4.64 (s, 1H), 4.22 (q, J=7.4 Hz, 1H), 4.05(td, J=7.8, 4.3 Hz, 1H), 3.47 (td, J=7.2, 4.3 Hz, 2H), 2.42-2.35 (m,2H), 2.28-2.16 (m, 2H), 1.75 (s, 9H), 1.68 (s, 6H) and 1.31 (t, J=7.3Hz, 3H) ppm.

EXAMPLE 25 Preparation ofBoc-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]ureadibenzyl phosphate (26)

ToBoc-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(25) (12.69 g, 23.58 mmol) and tetrazole (3.304 g, 47.16 mmol) under N₂at 23° C. was added DCM (240 mL) followed byN-dibenzyloxyphosphanyl-N-isopropyl-propan-2-amine (9.775 g, 9.509 mL,28.30 mmol). After 3 hours at 23° C., the reaction was cooled to 0° C.then mCPBA (6.977 g, 28.30 mmol) was added. The resulting solution wasstirred for 45 min at 0° C. then for 20 min at 23° C. The reactionmixture was then partitioned between DCM (50 mL) and saturated aqueoussodium bicarbonate (400 mL). The organic layer was separated, thenwashed successively with aqueous sodium bisulfite (63 g in 350 mL water)and saturated aqueous sodium bicarbonate (400 mL), then dried overmagnesium sulfate, filtered and concentrated in vacuo. The residue waspurified by MPLC using an ISCO COMBIFLASH brand flash chromatographypurification system (330 g silica column) eluting with a 0-100% EtOAc inhexanes linear gradient over 16 column volumes at 200 mL/min. Productcontaining fractions were evaporated in vacuo to giveBoc-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]ureadibenzyl phosphate (26) (11.92 g, 15.11 mmol, 64.09%). ESMS (M+1)=789.2;¹H NMR (300.0 MHz, CDCl₃) δ 9.51 (s, 1H), 9.03 (t, J=5.4 Hz, 1H), 8.91(d, J=1.6 Hz, 2H), 7.73 (d, J=6.5 Hz, 1H), 7.37-7.28 (m, 10H), 5.58 (t,J=7.8 Hz, 1H), 5.17-5.05 (m, 4H), 4.23 (t, J=7.5 Hz, 1H), 4.05 (td,J=7.8, 4.3 Hz, 1H), 3.53-3.44 (m, 2H), 2.39 (dd, J=7.9, 14.5 Hz, 2H),2.28-2.15 (m, 2H), 1.98 (s, 6H), 1.72 (m, 9H) and 1.31 (t, J=7.2 Hz, 3H)ppm.

EXAMPLE 26 Preparation of (R)-dibenzyl2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-ylphosphate (24)

To a solution ofBoc-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]ureadibenzyl phosphate (26) (11.9 g, 15.09 mmol) in DCM (300 mL) at 23° C.was added water (2.325 mL, 129.1 mmol) then TFA (3.441 g, 2.325 mL,30.18 mmol). After 1 h, only partial conversion was observed by tlc, somore TFA (3.441 g, 2.325 mL, 30.18 mmol) was added. After a further 2.5h, MeOH (2 mL) was added and the mixture stirred a further 18 hours. Thereaction mixture was washed with 1:1 brine:saturated aqueous sodiumbicarbonate (200 mL). The aqueous layer was re-extracted with DCM (150mL), the organic layers combined, then dried (over magnesium sulfate),filtered and concentrated in vacuo. The resulting residue wasre-dissolved in EtOAc (200 mL) washed with water (150 mL) and brine (100mL), then dried (magnesium sulfate) filtered and concentrated to give(R)-dibenzyl2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-ylphosphate (24) (10.21 g, 14.83 mmol, 98.27%) as a white solid. ESMS(M+1)=689.4; ¹H NMR (300 MHz, CD₃OD) δ 8.88 (d, J=1.6 Hz, 2H), 7.37 (d,J=6 Hz, 1H), 7.30 (m, 10H), 5.38-5.33 (m, 1H), 5.12-5.01 (m, 4H), 4.24(dd, J=6.8, 14.9 Hz, 1H), 3.98 (dd, J=6.9, 15.1 Hz, 1H), 3.35-3.27 (m,3H), 2.52 (q, J=5.9 Hz, 1H), 2.14-2.05 (m, 2H), 1.91 (s, 6H) and1.22-1.14 (m, 3H) ppm.

EXAMPLE 27 Preparation of disodium(R)-2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-ylphosphate (W)

A 1 L round bottom flask was charged with (R)-dibenzyl2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-ylphosphate (24) (9.37 g, 13.61 mmol), EtOH (300 mL), water (150 mL), Pd/C(10 wt % dry basis, wet, Degussa type, 3 g) and 1 M aqueous NaOH (27.22mL, 27.22 mmol). The suspension was evacuated for 3 min (needle to pump)then placed under an atmosphere of hydrogen gas (balloon). Afterstirring 2.5 h at 23° C., the reaction was filtered through a 0.22 μmPES membrane (disposable Corning filter system, 1 L, polystyrene,#431098) to remove catalyst and washed with EtOH (50 mL). The resultingsolution was concentrated, the residue dissolved in water (80 mL),treated with MeCN (80 mL), then frozen and lyophilized to give disodium(R)-2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-ylphosphate (W) (7.10 g, 12.81 mmol, 94.12%) as a white solid. ESMS(M+1)=509.3; ¹H NMR (300 MHz, D₂O) δ 8.58 (s, 2H), 6.92 (d, J=6.3 Hz,1H), 5.13 (t, J=7.5 Hz, 1H), 3.98-3.81 (m, 2H), 3.04 (q, J=7.2 Hz, 2H),2.26 (t, J=5.7 Hz, 1H), 1.97-1.92 (m, 2H), 1.67 (s, 6H) and 1.01 (t,J=7.2 Hz, 3H) ppm.

EXAMPLE 28 Preparation ofdiBoc-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(28)

To a solution/suspension of1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(23) (1.333 g, 1111 mmol) in DMF (30 mL) was added DMAP (38.01 mg,0.3111 mmol) followed by Boc₂O (1.426 g, 1.501 mL, 6.533 mmol). After 30min, the reaction mixture was diluted with water and EtOAc (300 mLeach), the organic layer separated, washed with water and brine (300 mLeach), then dried over magnesium sulfate, filtered and concentrated. Theresidue was purified by MPLC using an ISCO COMBIFLASH brand flashchromatography purification system (80 g silica column) eluting with a0-60% EtOAc in hexanes linear gradient over 20 column volumes at 60mL/min flow rate. Desired product fractions were combined and evaporatedto givediBoc-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(28) (1.43 g, 2.275 mmol, 73.11%) as a clear foam. ESMS (M+1)=629.3; ¹HNMR (300.0 MHz, CDCl₃) δ 8.95 (d, J=1.6 Hz, 2H), 8.31-8.27 (m, 1H), 8.05(d, J=6.5 Hz, 1H), 5.80-5.68 (m, 1H), 4.70 (s, 1H), 4.21-4.09 (m, 1H),3.98 (d, J=6.4 Hz, 1H), 3.42-3.37 (m, 2H), 2.45-2.00 (m, 4H), 1.65 (s,6H), 1.62 (s, 9H), 1.37 (s, 9H) and 1.28-1.21 (m, 3H) ppm.

EXAMPLE 29 Preparation ofdiBoc-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]ureadibenzyl phosphate (29)

TodiBoc-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]urea(28) (1.13 g, 1.797 mmol) and tetrazole (251.8 mg, 3.594 mmol) at 23° C.under N₂ was added DCM (30 mL) followed byN-dibenzyloxyphosphanyl-N-isopropyl-propan-2-amine (744.7 mg, 724.4 μL,2.156 mmol). After stirring for 18 h, the reaction was cooled to 0° C.then treated with mCPBA (531.5 mg, 2.156 mmol). The reaction was stirredfor 15 min at 0° C., then for 30 min at 23° C. The resulting solutionwas then partitioned between EtOAc and saturated aqueous sodiumbicarbonate (300 mL each), the organic layer separated, then washed with10% aqueous sodium bisulfite, saturated aqueous sodium bicarbonate andbrine (300 mL each), then dried over magnesium sulfate filtered andconcentrated. The residue was purified by MPLC using an ISCO COMBIFLASHbrand flash chromatography purification system (80 g silica column)eluting with a 0-80% EtOAc in hexanes linear gradient over 20 columnvolumes at 60 mL/min flow rate. Desired product fractions were combinedand evaporated to givediBoc-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]ureadibenzyl phosphate (29) (1.03 g, 1.159 mmol, 64.50%) as a clear, glassyoil. ESMS (M+1)=889.5; ¹H NMR (300.0 MHz, CDCl₃) δ 8.93 (d, J=1.5 Hz,2H), 8.31 (s, 1H), 8.04 (d, J=6.4 Hz, 1H), 7.36-7.26 (m, 10H), 5.83-5.70(m, 1H), 5.16-5.05 (m, 4H), 4.24-4.18 (m, 1H), 4.03-3.97 (m, 1H),3.42-3.36 (m, 2H), 2.43-2.05 (m, 4H), 1.98 (s, 6H), 1.64 (s, 9H), 1.40(s, 9H) and 1.26 (t, J=7.2 Hz, 3H) ppm

EXAMPLE 30 Preparation of sodium(R)-2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-ylphosphate (W)

To a solution ofdiBoc-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]ureadibenzyl phosphate (29) (121 mg, 0.1361 mmol) in DCM (10 mL) at 23° C.was added TFA (5 mL). After 2 h, the reaction mixture was concentratedin vacuo. The residue was dissolved in MeOH (6 mL) and treated withapprox 0.5 mL 2 M NH₃ in MeOH (to fully dissolve the material). Theresulting solution was purified in 6 injections on preparative HPLC,reverse phase, Sunfire prep C18 OBD 5 μM column 19×100 mm; eluting witha 10-90% aq MeCN w/0.1% TFA buffer, linear gradient over 15 min at 20mL/min flow rate. Fractions containing product were pooled andlyophilized. The resulting material was suspended in MeOH (3 mL),stirred at 23° C. for 30 min, then the precipitate was collected viafiltration through a plastic frit. The resulting white solid wasre-subjected to a MeOH slurry (3 mL), then collected via filtration togive 68 mg of white solid after drying. The white solid was treated with0.10 M aq NaOH (2.68 mL, 2 equiv NaOH) to give a solution that was thenpassed through an Acrodisc CR 13 mm syringe filter with 0.45 μm PTFEmembrane, flushing with water (2 mL). The resulting solution was treatedwith MeCN (3 mL), frozen and lyophilized to give sodium(R)-2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-ylphosphate (W) as a white powder. ESMS (M+1)=509.2; ¹H NMR (300 MHz, D₂O)δ 8.58 (s, 2H), 6.92 (d, J=6.3 Hz, 1H), 5.13 (t, J=7.5 Hz, 1H),3.98-3.81 (m, 2H), 3.04 (q, J=7.2 Hz, 2H), 2.26 (t, J=5.7 Hz, 1H),197-1.92 (m, 2H), 167 (s, 6H) and 1.01 (t, J=7.2 Hz, 3H) ppm.

EXAMPLE 31 Susceptibility Testing in Liquid Media

Compounds of this invention were tested for antimicrobial activity bysusceptibility testing in liquid media. Such assays were performedwithin the guidelines of the latest CLSI document governing suchpractices: “M07-A8 Methods for Dilution Antimicrobial SusceptibilityTests for Bacteria that Grow Aerobically; Approved Standard—EighthEdition (2009)”. Other publications such as “Antibiotics in LaboratoryMedicine” (Edited by V. Lorian, Publishers Williams and Wilkins, 1996)provide essential practical techniques in laboratory antibiotic testing.The specific protocols used were as follows:

Protocol 4. MIC Determination Procedure for Mycobacterium Species

Materials

Round bottom 96-well microtiter plates (Costar 3788) or similar

Film plate seals (PerkinElmer, TopSeal-A #6005250 or similar)

Middlebrook 7H10 broth with 0.2% glycerol

Middlebrook 7H10 agar with 0.2% glycerol

Middlebrook OADC Enrichment

Inoculum Preparation for M. tuberculosis:

-   -   1. Used prepared frozen M. tuberculosis stock stored at        −70° C. M. tuberculosis was grown in 7H10 broth+10% OADC, then        frozen at a concentration of 100 Klett or 5×10⁷ cfu/ml,    -   2. Prepared a 1:20 dilution by removal of 1 ml of the frozen        stock and added it to 19 ml of 7H10 broth+10% OADC (final        concentration 2.5×10⁶ cfu/ml).    -   3. From this dilution prepared a second 1:20 dilution, removed 1        ml and added it to 19 ml of fresh broth. This was the final        inoculum to add to the 96-well plates.

Inoculum Preparation for M. kansasii, M. avium, M. abscessus andNocardia spc.:

-   -   1. Used prepared frozen stock of culture or a fresh culture        grown in 7H10 broth at a concentration of 10 Klett or 5×10⁷/ml.    -   2. Prepared a 1:20 dilution by removing 1.0 ml of the culture        stock and added it to 19 ml of 7H10 broth (final concentration        2.5×10⁶ cfu/ml).    -   3. From this dilution prepared a 1:20 dilution, removed 1 ml and        added it to 19 ml of fresh broth (final suspension).

Plate Preparation:

-   -   1. Labeled plates.    -   2. Added 50 μl of 7H10 broth+10% OADC to all wells being        utilized for MIC determination using a multichannel electronic        pipettor.    -   3. Prepared stock solutions of drugs (e.g. 1 mg/ml        concentration) to be tested.    -   4. Thawed and diluted frozen stock solutions using 7H10        broth+10% OADC to obtain a working solution 4× the maximum        concentration tested (e.g. final concentration 32 μg/ml, highest        concentration tested was 8 μg/ml). Dilutions were made from the        stock solution. To start at a concentration of 1 μg/ml, the        drugs were prepared at 4 μg/ml, so the starting concentration        was 1 μg/ml. Removed 25 μl of the 1 mg/ml stock and added to 6.2        ml of broth. All dilutions of drugs were done in broth.    -   5. Added 50 μl of the 4× working solution to the first well of        the designated row. Continued for all compounds to be tested.        Using a multichannel electronic pipettor, mixed 4× and serial        diluted compounds through the 11th well. Discarded remaining 50        μl. Used the 12th well as the positive control.    -   6. Incubated plates at 37° C. M. tuberculosis for ˜18 days; M.        avium and M. kansasii for ˜7 days; Nocardia and M. abcessus for        ˜4 days; with film seals.    -   7. Read visually and recorded the results. The MIC was recorded        as the lowest concentration of drug where no growth was observed        (optical clarity in the well).

Protocol 5. Protocol for Mycobacterium tuberculosis Serum Shift MICAssay

Materials and reagents:

Costar #3904 Black-sided, flat-bottom 96-well microtiter plates

Middlebrook 7H9 broth (BD271310) with 0.2% glycerol

Middlebrook OADC Enrichment

Fetal Bovine Serum

Catalase (Sigma C1345)

Dextrose

NaCl₂

BBL Prompt Inoculation System (Fisher b26306)

Agar plates (Middlebrook 7H11 with 0.2% glycerol and OADC enrichment)with bacteria streaked to single colonies

Sterile DMSO

Media Prep:

-   -   1. For serum shifted MICs, three different media were required        which all had a base of 7H9+0.2% glycerol. It was important that        all media and supplements were sterilized prior to MICs.    -   2. Prepared all media below and inoculated as described in next        section. Tested all compounds against Mtb using each media.        -   a. 7H9+0.2% glycerol+10% OADC (“standard” MIC media).        -   b. 7H9+0.2% glycerol+2 g/L dextrose+0.85 g/L NaCl+0.003 g/L            catalase (0% FBS).        -   c. 2×7H9+0.2% glycerol+2 g/L dextrose+0.85 g/L NaCl+0.003            g/L catalase combined with equal volume Fetal Bovine Serum            (50% FBS).

Inoculum Prep:

-   -   1. Using BBL Prompt, picked 5-10 well-separated colonies and        inoculated 1 ml sterile saline that came in the kit. Typically        plates were two to three weeks of age when used for this assay        due to the slow growth of this organism in culture.    -   2. Vortexed well, then sonicated in water bath for 30 sec        providing a suspension of ˜10⁸ cells/ml. Actual density could be        confirmed by plating out dilutions of this suspension.    -   3. Prepared inoculum in each of the three media formulations by        diluting the BBL Prompt suspension 1/200 (for example:        transferred 0.2 ml of cells to 40 ml of medium) to obtain a        starting cell density of ˜10⁶ cells/ml.    -   4. Used 100 μl cells (˜5×10⁴ cells) to inoculate each microtiter        well containing 1 μl of drug in DMSO (see below).

Drug Dilutions, Inoculation, MIC Determination:

-   -   1. Control drug stocks Isoniazid and Novobiocin were prepared at        10 mM in 100% DMSO while Ciprofloxacin and Rifampin were        prepared at 1 mM in 50% DMSO and 100% DMSO, respectively.        Prepared dilutions-dispensed 100 μL of the stock solution into        the first column of a 96-well plate. Prepared 11-step, 2-fold        serial dilutions across the row for each compound by        transferring 50 μl from column 1 into 50 μl of DMSO in column 2.        Continued to transfer 50 μL from column 2 through column 11        while mixing and changing tips at each column. Left column 12        with DMSO only as a control.    -   2. Transferred 1 μl of each dilution into an empty microtiter        well prior to the addition of 100 μl of cells. The starting        concentration of Isoniazid and Novobiocin was 100 μM after the        dilution into medium+cells; the starting concentration of        Ciprofloxacin and Rifampin was 10 μM after the dilution into        medium+cells. Compound concentrations decreased in 2× steps        moving across the rows of the microtiter plate. All MICs were        done in duplicate at each of the three medium conditions.    -   3. Test sets of compounds were typically at 10 mM and 50 μL        volume.    -   4. Used a multichannel pipettor, removed all of the volume from        each column of the master plate and transferred into the first        column of a new 96-well microtiter plate. Repeated for each        column of compounds on master plate, transferring into column 1        of a new 96-well plate.    -   5. As described above for control compounds, generated 2-fold,        11-point dilutions of each compound using DMSO as diluent. In        all cases, left column 12 as DMSO only for a control. Once all        dilutions were complete, again transferred 1 μl of each dilution        into an empty microtiter well prior to the addition of 100 μl of        cells as done for the control compounds.    -   6. All wells were inoculated with 100 μl of diluted cell        suspension (see above).    -   7. After inoculum addition, mixed plates by gently tapping sides        of plate.    -   8. Plates were incubated in a humidified 37° C. chamber for 9        days.    -   9. At 9 days added 25 μl 0.01% sterile resazurin to each well.        Measured background fluorescence at Excitation 492 nm, Emission        595 nm and returned the plate to the incubator for another 24        hours.

After 24 hours the fluorescence of each well was measured at Excitation492 nm, Emission 595 nm.

Percent inhibition by a given compound was calculated as follows:Percent inhibition=100−([well fluorescence-average backgroundfluorescence]/[DMSO control−average background fluorescence]×100). MICswere scored for all three medium conditions as the lowest compoundconcentration that inhibited resazurin reduction (‘%−inhibition’) signal≧70% at a given medium condition.

Table 7 shows the results of the MIC assay for selected compounds ofthis invention.

In Table 7 and in subsequent Tables and Examples, “Compound 12”corresponds to1-ethyl-3-[5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]ureaand “Compound 13” relates to the mesylate salt of Compound 12.Similarly, “Compound 23” corresponds to1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl)pyrimidin-5-yl]-7-[(2R)-tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]ureaand “Compound 23A” relates to the mesylate salt of Compound 23. Theseare the same numbers used to identify said compounds and salts as usedin the Examples above.

TABLE 7 MIC Values of Selected Compounds MIC (μg/ml) Compound CompoundStrain/Special Condition Protocol 13 23A Mycobacterium avium 103 4 0.470.18 M. avium Far 4 0.94 0.23 M. avium 3404.4 4 0.94 0.23 M. kansasii303 4 Not Done 0.03 M. kansasii 316 4 Not Done 0.06 M. kansasii 379 4Not Done <0.015 M. tuberculosis H37Rv 4 0.37 0.015 ATCC 25618 M.tuberculosis Erdman 4 0.25 0.06 ATCC 35801 M. tuberculosis Erdman 50.045 0.03 ATCC 35801 M. tuberculosis Erdman 5 2 0.5 ATCC 35801 withMouse Serum M. abscessus BB2 4 Not Done 1 M. abscessus MC 6005 4 NotDone 1 M. abscessus MC 5931 4 Not Done 0.5 M. abscessus MC 5605 4 NotDone 1.5 M. abscessus MC 6025 4 Not Done 0.75 M. abscessus MC 5908 4 NotDone 1.5 M. abscessus BB3 4 Not Done 0.5 M. abscessus BB4 4 Not Done 2M. abscessus BB5 4 Not Done 0.5 M. abscessus MC 5922 4 Not Done 0.25 M.abscessus MC 5960 4 Not Done 0.5 M. abscessus BB1 4 Not Done 2 M.abscessus MC 5812 4 Not Done 1 M. abscessus MC 5901 4 Not Done 1 M.abscessus BB6 4 Not Done 0.5 M. abscessus BB8 4 Not Done 0.5 M.abscessus MC 5908 4 Not Done 1 M. abscessus LT 949 4 Not Done 1 M.abscessus BB10 4 Not Done 0.015 M. abscessus MC 6142 4 Not Done 0.5 M.abscessus MC 6136 4 Not Done 0.5 M. abscessus MC 6111 4 Not Done 0.5 M.abscessus MC 6153 4 Not Done 1

EXAMPLE 32

Seven-Day Oral (Gavage) Toxicity and Toxicokinetics Study in Rats

The objectives of this study were: 1) to evaluate the potential toxicityof Compound 13 and Compound 23A when administered orally by gavage tomale rats for 7 consecutive days and 2) to assess the toxicokinetics ofCompound 13, and Compound 23A after the first and seventh doses.

Animals

Species, Source, History, and Justification

Crl:CD(SD) rats were obtained from Charles River Laboratories of StoneRidge, N.Y. The animals were laboratory bred and experimentally naïve.Rats were chosen because they are a species that is commonly used fornonclinical toxicity evaluations.

Number, Sex, Age, and BodyWeightRange

Forty rats (20 noncannulated males and 20 males with jugular veincannulas) were ordered. From these animals, 15 noncannulated males and15 cannulated males were used. Animals were as uniform in age aspossible. The rats were prepubertal to young adult, approximately 9weeks of age at initiation of dosing. Their supplier-calculated birthdate was retained in the study records. The weight range for the animalsat the time of allocation to groups was 218.5-306.3 g.

Study Design

The rats were assigned as shown in the Table 8 below. Animals receivedthe test article or vehicle by oral gavage for 7 consecutive days andwere terminated the day following completion of dosing. The first day ofdosing was designated as Day 1 of the study. The animals were evaluatedfor changes in clinical signs, body weight, and other parameters asdescribed below.

TABLE 8 Group Assignment and Dose Levels No. Animals No. Animals DoseDose Dose Animals for Dose (M) (M) Test Level Doses Concentration VolumeNecropsy Group Main Study Toxicokinetics Article (mg/kg/day) per Day(mg/mL) (mL/kg) (Day 8) A 3 0 Vehicle 0 1 0 10 3 B 3 3 Compound 100 1 1010 6 13 C 3 3 Compound 200 1 20 10 6 13 D 3 3 Compound 100 1 10 10 6 23AE 3 3 Compound 300 2 30 10 6 23A F 0 3 Vehicle 0 2 0 10 3

Route/Dose

The vehicle and test article were administered by oral gavage once dailyfor 7 consecutive days at a dose volume of 10 mL/kg body weight forGroup A and Groups B-D, respectively. The test article and vehicle wereadministered by oral gavage twice daily, approximately 8 hours apart,for 7 consecutive days at a dose volume of 10 mL/kg body weight forGroup E and Group F, respectively. The actual volume administered toeach animal was calculated and adjusted based on the most recent bodyweight of each animal.

In-Life Observations and Measurements

Observations

Animals were observed for viability at least once in the morning andonce in the afternoon, at least 4 hours apart, throughout the study.During the treatment period, daily cageside observations were made andrecorded predose and postdose (following the first dose only). Thepostdosing observations made during treatment occurred at the followingtimes based on C_(max)/T_(max) for the two compounds from previousstudies:

1 hour postdose for Groups A-F.

One cageside observation was made on the day of necropsy.

Unscheduled Observations

Any findings observed at times other than scheduled observation timeswere to be recorded on an unscheduled observation or in Provantis;however, no abnormalities were observed throughout the study. Provantisis an electronic data collection, management and reporting system thatis commonly used in the art.

Body Weights

Prior to start of dosing, body weights were measured for randomizationon Day 1. During the treatment, body weights were measured on Day 1 andDay 7. In addition, fasted body weights were measured prior to necropsyfor calculation of organ/body weight ratios.

Food Consumption

Throughout the study, food consumption was measured daily starting 3days prior to start of dosing.

Clinical Pathology Evaluation

Blood samples for evaluation of hematology, coagulation, and serumchemistry parameters were collected from all animals from theretro-orbital plexus (under CO₂/O₂ anesthesia, for the main studyanimals) or jugular vein cannula (for the toxicokinetic animals) priorto necropsy. Due to residual heparin used to keep the cannulas patentfor the toxicokinetic animals, coagulation samples from these rats, werenot able to be analyzed. The animals were fasted overnight prior toblood collection. On the day of blood collection for clinical pathologyanalyses, the animals were not necropsied until after the blood wascollected and the samples judged to be acceptable by the clinicalpathology group.

Hematology

An appropriate amount of blood was collected in EDTA-containing tubes.The whole blood samples were analyzed for the parameters indicated belowin Table 9.

TABLE 9 Whole Blood Parameters Red blood cells (RBC) Mean corpuscularvolume (count and morphology) (MCV) White blood cells (WBC) Meancorpuscular hemoglobin (total and differential) (MCH) Hemoglobinconcentration Mean corpuscular hemoglobin (HGB) concentration (MCHC)Hematocrit (HCT) Platelet count (PLAT) Reticulocyte count Mean plateletvolume (ABSRET) (MPV)

Coagulation

An appropriate amount of blood was collected in tubes containing sodiumcitrate and then centrifuged to obtain plasma for the determination ofprothrombin time (PT) and activated partial thromboplastin time (APTT).

Serum Chemistry

An appropriate amount of blood was collected in tubes withoutanticoagulant. The sample was allowed to clot and then was centrifugedto obtain serum. The serum was analyzed for the parameters indicatedbelow in Table 10.

TABLE 10 Serum Parameters Sodium (NA) Calcium (CA) Potassium (K)Inorganic phosphorus (PHOS) Chloride (CL) Glucose (GLU) Total bilirubin(TBILI) Urea nitrogen (BUN) Alkaline phosphatase (ALKP) Total protein(TPRO) Lactate dehydrogenase (LDH) Albumin (ALB) Aspartateaminotransferase (AST) Globulin (GLOB) Alanine aminotransferase (ALT)Albumin/globulin ratio (A/G) Gamma-glutamyltransferase (GGT) Cholesterol(CHOL) Creatine phosphokinase (CK) Triglycerides (TRIG) Creatinine(CREA)

Toxicokinetics

On the 1^(st) and 7^(th) day of dosing, blood samples (approximately 0.5mL/sample) were collected from the jugular vein cannula for alltoxicokinetic animals at the timepoints listed below intoK₃EDTA-containing tubes. Toxicokinetic animals from the control group(Group F) only had a single blood collection sampling from eachcollection day at the 1-hour timepoint (following the first doseadministration of the day). Prior to each collection, a small sample ofblood (with heparin blocking solution) was removed from the cannula anddiscarded. A new syringe was placed on the cannula, and theprotocol-required sample was taken. The syringe with the blood samplewas removed, and a new syringe with saline attached to the cannula.Blood volume was replaced with an equal volume of saline and thenblocking solution placed in the cannula. Each animal was returned to itscage until the next collection timepoint.

All samples collected during this study were placed in labeledcontainers. Each label contained the following information: 1) Studynumber, 2) Animal number, 3) collection interval, 4) Group and Sex, and5) Date of collection.

The blood samples were mixed immediately by inverting, then placed onwet ice and centrifuged cold (˜1500 g, ˜10 minutes, ˜5° C.) to obtainplasma. The plasma was split into 96-well 1.4-mL polypropylene tubeswith pierceable TPE capcluster certified RNase, DNase free caps as2aliquots and stored frozen (≦−70° C.).

TABLE 11 Sample Collection Timepoints Timepoint Window¹ Predose Predose 1 h  ±4 min  2 h² ±5 min  4 h  ±5 min  8 h³ ±5 min 12 h  ±10 min  24 h ±20 min  48 h⁴ ±40 min  ¹All samples were collected within thecollection window. ²Following Day 1 dosing only. ³Obtained from Groups Eand F prior to PM dose administration. ⁴Following Day 7 dosing only.

Termination

No animal was deemed moribund during the study. All study animals wereeuthanized and subjected to a necropsy following the protocol-prescribednumber of days of treatment. All animals were terminated byexsanguination (severing the abdominal aorta while under deep CO₂/O₂anesthesia).

Necropsy

A necropsy with tissue collection was conducted on all animalsterminated during the study. The necropsy included examination of:carcass and muscular/skeletal system; all external surfaces andorifices; cranial cavity and external surface of the brain; neck withassociated organs and tissues; and thoracic, abdominal, and pelviccavities with their associated organs and tissues.

All abnormalities were described and recorded.

Organ Weights

For all animals euthanized at scheduled necropsies, the kidneys, liver,and prostate gland were weighed. Following weighing, an approximate 1gram sample of liver and kidney was weighed, transferred to Precellys 7mL CK28 Tissue Homogenizing tubes (Cat. No. 0904-01), snap-frozen, andanalyzed.

Organ/body ratios were calculated using the terminal fasted body weightobtained prior to necropsy.

Tissue Preservation and Bone Marrow Collection

The tissues and organs indicated below in Table 12 were collected fromall animals and were preserved in 10% neutral-buffered formalin with theexception of the testes, epididymides, and eyes. Testes, epididymides,and eyes with optic nerve attached were fixed in Modified Davidson'sSolution for ˜24-48 hours, rinsed with water, and then transferred to10% neutral-buffered formalin for storage.

TABLE 12 Tissue Collection Submitted at Tissue Necropsy Organ WeightHistopathology Animal ID X Adrenal gland (2) X Aorta X Artery,mesenteric X Bone (femur) X Bone marrow (sternum) X Brain X Epididymides(2) X Esophagus X Eyes (2) X Gross lesions X Heart X Intestine, cecum XIntestine, colon X Intestine, duodenum X Intestine, jejunum X Intestine,ileum X Intestine, rectum X Kidneys (2) X X X Liver X X X Lungs X Lymphnode, mandibular X Lymph node, mesenteric X Mammary gland X Nerve, opticX Nerve, sciatic X Parathyroid gland (2)^(a) X Pancreas X Pituitary XProstate X X X Seminal vesicles X Skeletal muscle (biceps X femoris)Skin (abdominal) X Spinal cord, cervical X Spinal cord, thoracic XSpinal cord, lumbar X Spleen X Stomach X Testes (2) X Thymus X Thyroidgland (2)^(a) X Tongue X Trachea X Urinary bladder X ^(a)Thyroid weighedwith parathyroids attached.

Histopathology

For all animals scheduled for the terminal necropsy, the kidneys, liver,and prostate were embedded in paraffin, sectioned and stained withhematoxylin and eosin for further examination by light microscopy. ForGroups A, D, E, and F only, the remaining tissues listed above wereembedded in paraffin, sectioned and stained with hematoxylin and eosinfor further examination by light microscopy.

Statistical Analysis

Where appropriate, numeric animal data were evaluated statistically.

For comparative statistics, Group A (control group) was compared toGroups B and C (treated groups, dosed QD) and Group F (control group,dosed BID) was compared to Group E (treated group, dosed BID). Data wereevaluated using the Levene Test for homogeneity of variances and theShapiro-Wilks Test for normality of distributions, with significance atp≦0.05. Data determined to be homogeneous and of normal distributionwere evaluated by analysis of variance (ANOVA). If the ANOVA verifiedsignificance at p≦0.05, pairwise comparisons of each treated group withthe respective control group were made using a parametric test (DunnettTest) to identify statistical differences (p≦0.05). Data determined tobe nonhomogeneous or of nonnormal distribution were evaluated using aKruskal-Wallis Test for group factor significance. If significance(p≦0.05) existed between groups, a nonparametric test (WilcoxonwithBonferroni-Holm), was used to compare treatment groups to the controlgroup. Food consumption data from animals where spillage occurred wasexcluded from the applicable time period. Comparative statistics of foodconsumption data were limited to the Dunnett Test (parametric).Statistics were not performed on pretest food consumption (Day 4 to Day1).

Results

The exposures for different dosage levels of Compound 23A and Compound13 were dose related. No adverse observations or effects on mean bodyweight were observed in animals treated with either Compound 13 orCompound 23A. Mean food consumption was reduced during differentintervals of the study for animals treated once daily with Compound 13(100 or 200 mg/kg) and twice daily with Compound 23A (300 mg/kg).However, as the decreased food consumption was not correlated with bodyweight changes in the Compound 13 and Compound 23A groups, these effectswere not considered to be adverse or biologically significant. The meancalcium ion concentration (CA) was statistically lower, while the meanALT and the AST for the group of rats administered 300 mg/kg Compound23A twice a day were statistically higher when compared to the controlstreated twice a day. No test article-related histopathological findingswere noted for animals receiving either Compound 13 or Compound 23A atany dose regimen.

Within the scope of this study and based on the absence of changes inbody weight, clinical pathology, and histopathology, the NOEL(No-Observable-Effect-Level) for Compound 13 administered to male ratsonce a day for 7 days orally via gavage was 200 mg/kg (844 μg*hr/ml Day7 AUC), while the NOEL for Compound 23A administered once a day was 100mg/kg (82 μg*hr/ml AUC). The NOAEL (No-Observable-Adverse-Effect-Level)for Compound 23A administered to male rats twice a day for 7 days orallyvia gavage was 300 mg/kg (291 μg*hr/ml AUC).

Therefore, Compounds 13 and 23A did not demonstrate adverse toxicitywithin the scope of the study at dose levels up to 200 mg/kg/day and 600mg/kg/day, respectively.

EXAMPLE 33

An Oral Range Finding Toxicity and Toxicokinetic Study in MaleCynomolgus Monkeys

The objectives of this study were 1) to evaluate the potential toxicityof Compound 23 when administered orally by gavage to male Cynomolgusmonkeys for 7 consecutive days; and 2) to assess the toxicokinetics ofCompound 23 after the first and seventh doses.

Animals

Species, Source, History, and Justification

Cynomolgus monkeys (Macaca Fascicularis) were obtained from PrimusBio-Resources Inc. of PinCourt, Quebec, Canada. Cynomolgus monkeys werechosen because they are a non-rodent species that is commonly used fornonclinical toxicity evaluations.

Number, Sex, Age, and Body Weight Range

Eight (2 naive and 6 non-naïve) males were used in the study. Theanimals were young adults and weighed between 2 to 4 kg at the onset ofdosing.

Study Design

The animals were assigned as shown in Table 13 below. Animals receivedCompound 23 or vehicle by oral gavage once per day for 7 consecutivedays and were terminated the day following completion of dosing. Thefirst day of dosing was designated as Day 1 of the study. The actualvolume administered to each animal was calculated and adjusted based onthe most recent body weight of each animal.

TABLE 13 Group Assignment and Dose Levels Dose Dose Level ConcentrationDose Volume Number of Group (mg/kg) (mg/mL) (mL/kg) animals 1 Control* 05 2 2 50 10 5 2 3 100 20 5 2 4 200 40 5 2 *The Control animals receivedthe control/vehicle (20% captisol/1% HPMCAS/1% PVP in 0.01M KCl/HCLbuffer) alone

In-Life Observations and Measurements

Observations

Cage-side clinical signs (ill health, behavioral changes etc.) wererecorded at least once daily during the study.

Body Weights

Body weights were recorded for all animals prior to group assignment andon Days 1 (prior to dosing), 3 and 7 as well as terminally prior tonecropsy (fasted).

Electrocardiography (ECG)

Electrocardiograms (bipolar limb leads I, II and III, and augmentedunipolar leads aVR, aVL and aVF) were obtained for all monkeys onceduring the pre-treatment period and again on Day 7 (post-dosing).

The tracings were assessed for gross changes indicative of cardiacelectrical dysfunction. The potential presence of abnormalitiesinvolving heart rate (lead II), sinus and atrioventricular rhythm orconductivity were determined. Heart rate, PR interval, QRS duration, QTand QTc intervals values were measured.

Toxicokinetics

A series of 7 blood samples (approximately 0.5 mL each) were collectedfrom each monkey on Days 1 and 7 at the following time points: predose,30 minutes and 2, 3, 6, 12 and 24 hours post-dose. For this purpose,each monkey was bled by venipuncture and the samples were collected intotubes containing the anticoagulant, K2EDTA. Tubes were placed on wet iceuntil ready for processing.

Clinical Pathology

Laboratory investigations (hematology, coagulation, clinical chemistryand urinalysis) were performed on all animals prior to start oftreatment and prior to termination on Day 8.

Blood samples were collected by venipuncture following an overnightperiod of food deprivation consisting of at least 12 hours but no morethan 20 hours. Urine was collected from animals deprived of food andwater, overnight (at least 12 hours but no more than 20 hours).

Hematology

The following parameters were measured on blood samples collected intoEDTA anticoagulant: red blood cell count, mean corpuscular hemoglobin(calculated), hematocrit (calculated), mean corpuscular volume,hemoglobin, morphology of cells, white blood cell count, platelet count,white blood cell differential (absolute), reticulocyte (absolute andpercentage) and mean corpuscular hemoglobin concentration (calculated).

Coagulation

Activated partial thromboplastin time and prothrombin time were measuredon blood samples collected into citrate anticoagulant.

Clinical Chemistry

The following parameters were measured on blood samples collected intotubes containing clotting activator: a/g ratio (calculated), creatinine,alanine aminotransferase, globulin (calculated), albumin, glucose,alkaline phosphatase, phosphorus (inorganic), aspartateaminotransferase, potassium, bilirubin (total), sodium, calcium, totalprotein, chloride, triglycerides, cholesterol (total), urea, gammaglutamyltransferase and sorbitol dehydrogenase.

Urinalysis

The following parameters were measured on urine samples: bilirubin,protein, blood, sediment microscopy, color and appearance, specificgravity, glucose, urobilinogen, ketones, volume and pH.

Termination

All animals were euthanized upon completion of the treatment period onDay 8 following an overnight period without food. The monkeys werepre-anesthetized with Ketamine and then euthanized by an intravenousoverdose of sodium pentobarbital followed by exsanguination bytranssection of major blood vessels.

Necropsy

A necropsy with tissue collection was conducted on all animalsterminated during the study. The necropsy included examination of:

-   -   carcass and muscular/skeletal system;    -   all external surfaces and orifices;    -   cranial cavity and external surface of the brain;    -   neck with associated organs and tissues; and    -   thoracic, abdominal, and pelvic cavities with their associated        organs and tissues.        All abnormalities were described and recorded.

Tissue Preservation

On completion of the gross examination and selected organ weighing, thetissues and organs were retained as noted below in Table 14. Neutralbuffered 10% formalin was used for fixation and preservation unlessotherwise indicated.

TABLE 14 Tissue and Organ Retention Weigh Examine ORGANS/TISSUESRetain(•) (✓ ) (

 ) Adrenals • ✓

Animal identification • Aorta (thoracic) •

Blood Bone marrow smears (3) • Brain • ✓

Cecum •

Colon •

Epididymides  •d

Esophagus •

Eyes  •a

Femur & marrow •

Gallbladder •

Heart • ✓

Kidneys • ✓

Liver (2 lobes) • ✓

Lungs (2 lobes)  •b  ✓c

Lymph Node, mandibular •

Lymph Node, mesenteric •

Mammary gland (thoracic) •

Optic nerves  •a

Pancreas •

Pituitary • ✓

Prostate • ✓

Rectum •

Salivary Gland, mandibular •

Sciatic nerve •

Seminal vesicles •

Skeletal muscle •

Skin & subcutis (thoracic) •

Duodenum •

Jejunum •

Ileum •

Spinal Cord, cervical •

Spleen • ✓

Sternum & marrow •

Stomach •

Testes  •d ✓

Thymus • ✓

Thyroid gland/parathyroids • ✓

Tongue •

Trachae  •c

Urinary bladder •

Abnormal findings • aDavidson's fluid used for fixation and preservationbLungs were infused with 10% neutral buffered formalin used for fixationand preservation cLungs were weighed with trachea dBouin's fluid usedfor fixation and preservation

 Examined microscopically

Histopathology

For all animals, all tissues indicated above were embedded in paraffin,sectioned and stained with hematoxylin and eosin and examined by lightmicroscopy.

Results

The exposures for different dosage levels of Compound 23 were doserelated.

There were no clinical signs, or changes in body weights,electrocardiography parameters, clinical pathology parameters, or organweights that could be attributed to the administration of Compound 23 atdoses up to 200 mg/kg/day. Similarly, there were no macroscopic ormicroscopic findings that could clearly be attributed to theadministration of Compound 23 at doses up to 200 mg/kg/day. The noobserved effect level (NOEL) for Compound 23 in male Cynomolgus monkeyswas determined to be 200 mg/kg/day.

EXAMPLE 34

Pharmacokinetic Studies

The pharmacokinetic parameters of selected compounds of this inventionwere determined in the experiments described below. General analyticprocedures and specific experimental protocols were employed as follows:

General Analytic Procedures

The following general analytic procedures were employed in thepharmacokinetic experiments described below:

Sample Analysis. Concentrations of Compound 23 and Compound W in plasmawere determined using a high performance liquid chromatography/tandemmass spectrometry (HPLC/MS/MS) method. Before extraction, plasma sampleswere diluted using blank plasma 2-, 4-, 5-, or 10-fold, as necessary,depending on the dose level or formulation. Compound 23 and Compound Walong with the internal standard (1S) were extracted from (diluted)plasma, 100 μL each, by direct protein precipitation with acetonitrile(1:4 ratio of plasma/acetonitrile). After centrifugation, thesupernatant extract (10 μL) was injected onto the LC/MS/MS system. TheHPLC system included a Waters Xterra MS C18 column, 5 micron, 2.1 mmdiameter×50 mm long eluted with a gradient mobile phase consisting of0.1% formic acid in water or in acetonitrile.

The analytes were detected by MS/MS with Atmospheric Pressure ChemicalIonization (APCI) in the mode of multiple reaction monitoring (MRM). Thelower limit of quantitation (LLOQ) was 1, 2, 4, 5, 10, or 20 ng/mL,depending on the sample dilution factor. The linear range of the assaywas from 1 to 5000 ng/mL. The intra-day and inter-day assay accuracy waswithin 2% of the nominal values. The intra- and inter-day assayvariability was <10%.

Samples of the dose suspension formulation of Compound W were assayedwith an HPLC/UV method after 10-fold to 500- or 1000-fold of dilutionwith DMSO:acetonitrile:water (33:33:33) depending on the dose level orformulation. Samples of the dose solution formulation of Compound W wereassayed with an HPLC/UV method after 10-, 50-, 100 or 500-fold ofdilution with DMSO:water (50:50) depending on the dose level orformulation.

Pharmacokinetic Data Analysis. Plasma concentration-time profiles ofCompound 23 and Compound W were analyzed by noncompartmentalpharmacokinetic methods using WinNonlin® Professional Edition software,Version 5.1.1 (Pharsight Corporation, Mountain View, Calif.).

Key pharmacokinetic parameters including AUC_(all), AUC_(extrap),C_(max), t_(max), Cl_obs, Vss_obs and t_(1/2) were determined.

Statistical Data Analysis. Descriptive statistical data of plasmaconcentrations and pharmacokinetic parameter estimates were calculated,including the mean, standard deviation (SD), and coefficient ofvariation (% CV) using WinNonlin software, Version 5.1.1 or MicrosoftExcel 2000.

Monkey Oral Study

Malecynomolgus monkeys (n=3 per dose group) were administered singlenominal PO doses of 3, 30 and 300 mg/kg of Compound W by gavage.Compound W was formulated in 0.5% MC (microcrystalline cellulose).Animals had free access to food and water before and after dosing.

Blood samples (approximately 0.25 mL each) were collected via a carotidartery catheter prior to dosing and at 0 (predose), 0.25, 0.5, 1, 2, 3,4, 6, 8, 12, 24, 48 hours post dose. Each blood sample was collectedinto a tube that was kept on wet ice and contained potassium EDTA as theanticoagulant. Plasma was separated and stored at approximately −70° C.until analysis.

Plasma samples were analyzed using a liquid chromatography/tandem massspectrometry (LC/MS/MS) method to determine the concentrations ofCompound 23 and Compound W with a lower limit of quantitation (LLOQ) of1 to 20 ng/mL, depending on the sample dilution factor. Plasmaconcentration vs. time data was subjected to noncompartmentalpharmacokinetic (PK) analysis. The results of this analysis are providedin Table 15.

TABLE 15 Pharmacokinetic Data from Monkey Oral Study Dose Cmax AUXAUCextrap Tmax t1/2 (mg/kg) Route Formulation Analyte (ug/ml) (ug *hr/ml) (ug * hr/ml) (hr) (hr) 30 PO 0.5% MC Compound 14.4 24.7 24.8 1.713.9 23 100 PO 0.5% MC Compound 20.9 76.7 76.9 2.3 8.3 23 300 PO 0.5% MCCompound 23.8 155.1 155 1.2 5.6 23 30 PO 0.5% MC Compound 0.0264 0.04530.206 0.83 — W 100 PO 0.5% MC Compound 0.322 0.432 0.437 0.67 5.31 W 300PO 0.5% MC Compound 4 3.69 3.76 0.58 13.15 W

Monkey IV Study

Male cynomolgus monkeys (n=3 per dose group) were administered a singlenominal IV bolus dose of 1 mg/kg of Compound W via a jugular veincannula. Compound W was formulated in D5W (5% dextrose in watersolution). Animals had free access to food and water before and afterdosing.

Blood samples (approximately 0.25 mL each) were collected via a carotidartery catheter prior to dosing and at 0 (predose), 5 min, 10 min, 0.25,0.5, 1, 2, 3, 4, 6, 8, 12, 24, 48 hours postdose. Each blood sample wascollected into a tube that was kept on wet ice and contained potassiumEDTA as the anticoagulant. Plasma was separated and stored atapproximately −70° C. until analysis.

Plasma samples were analyzed using a liquid chromatography/tandem massspectrometry (LC/MS/MS) method to determine the concentrations ofCompound 23 and Compound W, with a lower limit of quantitation (LLOQ) of1 to 20 ng/mL, depending on the sample dilution factor. Plasmaconcentration vs. time data were subjected to noncompartmentalpharmacokinetic (PK) analysis. The results of this analysis are providedin Table 16.

TABLE 16 Pharmacokinetic Data from Monkey IV Study C0 AUC Cl Dose (ug/(ug * hr/ AUCextrap (ml/min/ t1/2 Vss (mg/kg) Route Formulation Analyteml ml) (ug * hr/ml) kg) (hr) (L/kg) 5 IV D5W Compound 10.9 3.78 3.8123.4 6.17 2.09 23 5 IV D5W Compound 62.4 5.79 5.83 18.2 5.35 1.88 W

Rat Oral Study

Groups of maleSprague Dawley rats (n=3 per dose group) were administeredsingle nominal oral doses of 3, 10, 30, 300 mg/kg of Compound W bygavage. Compound W was formulated in either 0.5% MC (microcrystallinecellulose) or 20% Captisol, 1% HPMC-AS (hydroxypropyl methylcelluloseacetyl succinate), 1% PVP (polyvinylpyrrolidone). Animals had freeaccess to food and water before and after dosing. Blood samples(approximately 0.25 mL each) were collected via a carotid arterycatheter prior to dosing and at 0 (predose), 0.25, 0.5, 1, 1.5, 2, 4, 6,8, 12, 24 hours post dose. Each blood sample was collected into a tubethat was kept on wet ice and contained potassium EDTA as theanticoagulant. Plasma was separated and stored at approximately −70° C.until analysis.

Plasma samples were analyzed using a liquid chromatography/tandem massspectrometry (LC/MS/MS) method to determine the concentrations ofCompound 23 and Compound W with a lower limit of quantitation (LLOQ) of1 to 20 ng/mL, depending on the sample dilution factor. Plasmaconcentration vs. time data was subjected to noncompartmentalpharmacokinetic (PK) analysis. The results of this analysis are providedin Table 17.

TABLE 17 Pharmacokinetic Data from Rat Oral Study Drug Cmax/C0 AUCAUCextrap Tmax t1/2 (mg/kg) Formualtion Analyte (ug/ml) (ug * hr/ml)(ug * hr/ml) (hr) (hr) 3 0.5% MC Compound 0.117 0.311 0.314 0.58 4.06 2330 0.5% MC Compound 2.9 22.5 22.6 1.7 2.6 23 100 0.5% MC Compound 6.677.1 77.4 2.5 2.7 23 300 0.5% MC Compound 11.7 222.8 307.6 — 17.9 23 30020% CAPT, Compound 16.2 294.6 — 5 — 1% HPMC- 23 AS, 1% PVP 3 0.5% MCCompound — — — — — W 30 0.5% MC Compound 0.022 0.178 0.058 3.3 3.1 W 1000.5% MC Compound 0.021 0.061 0.066 0.8 7.2 W 300 0.5% MC Compound 2.330.324 0.464 1.2 11.3 W 300 20% CAPT, Compound 0.6 2.37 4.27 1.8 — 1%HPMC- W AS, 1% PVP

Rat IV Study

Groups of male Sprague Dawley rats (n=3 per dose group) wereadministered single nominal IV bolus doses of 1 and 5 mg/kg of CompoundW via a jugular vein cannula. Compound W was formulated in D5W. Animalshad free access to food and water before and after dosing. Blood samples(approximately 0.25 mL each) were collected via a carotid arterycatheter prior to dosing and at 0 (predose), 5 min, 10 min, 0.25, 0.5,1, 1.5, 2, 4, 6, 8, 12, 24 hours post dose. Each blood sample wascollected into a tube that was kept on wet ice and contained potassiumEDTA as the anticoagulant. Plasma was separated and stored atapproximately −70° C. until analysis.

Plasma samples were analyzed using a liquid chromatography/tandem massspectrometry (LC/MS/MS) method to determine the concentrations ofCompound 23 and Compound W with a lower limit of quantitation (LLOQ) of1 to 20 ng/mL, depending on the sample dilution factor. Plasmaconcentration vs. time data were subjected to noncompartmentalpharmacokinetic (PK) analysis. The results of this analysis are providedin Table 18.

TABLE 18 Pharmacokinetic Data from Rat IV Study Dose AUC (mg/ Cmax/C0(ug * hr/ AUCextrap t1/2 Cl_obs Vss_obs kg) Formualtion Analyte (ug/ml)ml) (ug * hr/ml) (hr) (ml/min/kg) (L/kg) 1 D5W Compound 0.247 0.306 0.311.8 54.9 3.8 23 5 D5W Compound 1.2 3.04 3.06 3.6 27.3 4.08 23 1 D5WCompound 4.8 0.416 0.419 0.9 46.7 0.38 W 5 D5W Compound 9.03 1.11 1.127.2 84.6 5.8 W

Mouse Oral Study

Groups of female CD-1 mice (n=3 per dose group) were administered singlenominal oral doses of 10, 30, 100 mg/kg of Compound W by gavage.Compound W was formulated in 0.5% MC. Animals had free access to foodand water before and after dosing. Blood samples (approximately 0.025 mLeach) were collected from the sub-mandibular vein prior to dosing and at0 (predose), 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24 hours postdose. Eachblood sample was collected into a tube that was kept on wet ice andcontained potassium EDTA as the anticoagulant. Plasma was separated andstored at approximately −70° C. until analysis.

Plasma samples were analyzed using a liquid chromatography/tandem massspectrometry (LC/MS/MS) method with a lower limit of quantitation (LLOQ)of 1 to 20 ng/mL, depending on the sample dilution factor. Plasmaconcentration vs. time data was subjected to noncompartmentalpharmacokinetic (PK) analysis. The results of this analysis are providedin Table 19.

TABLE 19 Pharmacokinetic Data from Mouse Oral Study Dose AUC (0-t) CmaxTmax (mg/kg) Formulation (μg*hr/mL) (μg*hr/ml) (hr) 10 0.5% MC 1.7 1.20.3 30 0.5% MC 4.1 2.1 0.3 100 0.5% MC 26.6 9.1 0.4

The studies described above, demonstrate that Compound W is converted invivo into Compound 23 in at least rats, dogs and monkeys.

EXAMPLE 35

Enzymology Studies

The enzyme inhibition activities of selected compounds of this inventionwere determined in the experiments described below:

DNA Gyrase ATPase Assay

The ATP hydrolysis activity of S. aureus DNA gyrase was measured bycoupling the production of ADP through pyruvate kinase/lactatedehydrogenase to the oxidation of NADH. This method has been describedpreviously (Tamura and Gellert, 1990, J. Biol. Chem., 265, 21342).

ATPase assays were carried out at 30° C. in buffered solutionscontaining 100 mM TRIS pH 7.6, 1.5 mM MgCl₂, 150 μM KCl. The couplingsystem contains final concentrations of 2.5 mM phosphoenol pyruvate, 200μM nicotinamide adenine dinucleotide (NADH), 1 mM DTT, 30 ug/ml pyruvatekinase, and 10 ug/ml lactate dehydrogenase. The enzyme (90 nM finalconcentration) and a DMSO solution (3% final concentration) of theselected compound were added. The reaction mixture was allowed toincubate for 10 minutes at 30° C. The reaction was initiated by theaddition of ATP to a final concentration of 0.9 mM, and the rate of NADHdisappearance was monitored at 340 nanometers over the course of 10minutes. The K_(i) and IC₅₀ values were determined from rate versusconcentration profiles.

Selected compounds of the present invention were found to inhibit S.aureus DNA gyrase. Table 20 shows the inhibitory activity of thesecompounds in the S. aureus DNA gyrase inhibition assay.

TABLE 20 Inhibition of S. aureus DNA Gyrase Selected Compound K_(i) (nM)IC₅₀ (nM) Compound 23 9 Compound W <9 54

DNA Topo IV ATPase Assay

The conversion of ATP to ADP by S. aureus TopoIV enzyme was coupled tothe conversion of NADH to NAD+, and the progress of the reaction wasmeasured by the change in absorbance at 340 nm. TopoIV (64 nM) wasincubated with the selected compound (3% DMSO final) in buffer for 10minutes at 30° C. The buffer consisted of 100 mM Tris 7.5, 1.5 mM MgCl₂,200 mM K.Glutamate, 2.5 mM phosphoenol pyruvate, 0.2 mM NADH, 1 mM DTT,5 μg/mL linearized DNA, 50 μg/mL BSA, 30 μg/mL pyruvate kinase, and 10μg/mL lactate dehyrodgenase (LDH). The reaction was initiated with ATP,and rates were monitored continuously for 20 minutes at 30° C. on aMolecular Devices SpectraMAX plate reader. The inhibition constant, Ki,and the IC₅₀ were determined from plots of rate vs. concentration ofselected compound fit to the Morrison Equation for tight bindinginhibitors.

Selected compounds of the present invention were found to inhibit S.aureus DNA Topo IV. Table 21 shows the inhibitory activity of thesecompounds in the S. aureus DNA gyrase inhibition assay.

TABLE 21 Inhibition of S. aureus DNA Topo IV Selected Compound K_(i)(nM) IC₅₀ (nM) Compound 23 12 Compound W 30 150

EXAMPLE 36

Aqueous Solubility Study

The aqueous solubilities of compound 23 and compound W were determinedaccording to the following procedure.

Preparation of Samples. Aqueous samples of each compound were preparedas follows. Compounds were weighed (20-30 mg compound) in 4 ml clearvials prior to adding water (0.5 mL) and stirring by magnetic stirrer.1.0N HCl was added to the suspension to adjust the pH to the desiredrange. After stirring for 96 hours at room temperature, the suspensionwas filtered through a 0.22 micron filter (Millipore, Ultrafreecentrifugal filters, Durapore PVDF 0.22 μm, Cat# UFC30GVNB). Thefiltrate was collected and the pH measured with a pH meter. The filtratecontaining compound W was diluted 10-fold to provide an appropriateconcentration for HPLC analysis. The filtrate containing compound 23 didnot require dilution.

Preparation of Standard Solutions. Standard solutions of each compoundwere prepared according to the following procedure. 1 to 2 mg of eachcompound was accurately weighed into a 10 mL volumetric flask and eitherwater (for compound W) or 1:1 methanol:0.1N HCl (for compound 23) wasadded to completely dissolve the compounds. Sonication was performed forcompound 23 to assist with the dissolution in 1:1 methanol:0.1N HCl.When all solids dissolved, additional solvent was added to adjust thevolume of each solution to 10 ml. The resulting solutions werethoroughly mixed to give the standard solutions of each compound. Eachstandard solution was then diluted with solvent by 2-fold, 10-fold, and100-fold.

Solubility Analysis. Aliquots of each sample and each standard solutionwere analyzed by HPLC analysis (Agilent 1100, injection volume 10 μL,wavelength 271 nm, column XTerra® Phenyl 5 μm, 4.6×50 mm, Part No.186001144, mobile phase: A: 0.1% TFA in water 0.1% TFA in AcN). Eachstandard solution was injected three times, and each of the samples wasinjected twice. Standard curves were obtained by plotting the average ofthe peak area from the HPLC versus the concentrations of the standardsolutions (with appropriate corrections of the weights of the standardsbased on total water content of the solid as determined by elementalanalysis). The concentration of each sample was calculated from the peakarea of the aqueous sample from the HPLC results and the slope andintercept of the standard curves. The solubility values listed in Table22 below were derived from the product of the concentration of thesample and the dilution factor of the sample.

TABLE 22 Aqueous Solubility of Compounds 23 and W Solubility CompoundSolid form pH (mg/mL) Compound 23 crystalline >3.0 <0.001 Compound Wcrystalline 4.39 0.25

EXAMPLE 37

In Vivo Metabolism Study 1N Hepatic and Liver S9 Cells

The conversion of Compound W to Compound 23 was studied in liver andintestinal S9 fractions from rats, dogs, monkeys and humans. Compound Wwas incubated at 0.1, 0.3, 1, 3, 10, 20, 40, 100, 200, 300 μM in liverS9 fractions and at 1, 3, 10, 20, 100, 300, 500, 1000 μM in intestinalS9 fractions. The incubations were done for 0, 5, 10, 15, 30, 45 or 60minutes. The formation of Compound 23 was quantified by LC/MS-MS anddata were fitted to the Michaelis Menten equation. The data in Table 23below indicates that Compound W rapidly converts to Compound 23 in thesehepatic and intestinal S9 fractions.

TABLE 23 Velocity of formation (V_(MAX)) of Compound 23 from Compound Win Liver and Intestinal S9 V_(MAX) (liver) V_(MAX) (intestine)(pmoles/min/mg) (pmoles/min/mg) Dog 19.3 1162 Monkey 25.2 1974 Rat 45.5958 Human 45.8 ND* *ND: Parameters not determined, rate of formation didnot saturate

EXAMPLE 38

Mouse M. tuberculosis (Erdman) Lung Infection Model)

Animals: female Balb/c mice (5-7 weeks of age; 6/group) were obtainedfrom Jackson Laboratories (Bar Harbor, Me.) and were housed andmaintained in a biosefatey level 3 (BSL3) facility in accordance withthe Guide to the Care and Use of Experimental Animals.

Bacterial Strain and Stocks

M. tuberculosis ATCC 35801 (strain Erdman) was obtained from the ATCC(Manassas, Va., USA). The organism was grown in 20 tubes of modified7H10 broth (pH 6.6; 7H10 agar formulation with agar and malachite greenomitted) with 10% OADC (oleic acid, albumin, dextrose, catalase)enrichment (BBL Microbiology Systems, Cockeysville, Md., USA) and 0.05%Tween 80 for 5-10 days on a rotary shaker at 37′C. The cultures werepooled and diluted to 100 Klett units [equivalent to 5×10⁷ colonyforming units (cfu)/mL] (Photoelectric Colorimeter; Manostat Corp., NewYork, N.Y., USA). The culture was aliquotted and frozen at −70° C. Onthe day of infection, the culture was thawed and the final inoculum wasdetermined. The final inoculum size was determined by diluting to 5×10⁻²and plating 0.1 mL, in triplicate, on 7H10 agar plates (BBL MicrobiologySystems) supplemented with 10% OADC enrichment. The plates wereincubated at 37′C in ambient air for 4 weeks.

Mouse M. tuberculosis (Erdman) Infection Model

For intranasal infection, groups of mice were anaesthetized byintramuscular delivery of a telazol (45 mg/kg)/xylazine (7.5 mg/kg)cocktail (Lederle Parenterals, Carolina, Puerto Rico and Bayer Corp.,Shawnee Mission, Kans., USA, respectively) and subsequently infectedintranasally with ˜10² viable M. tuberculosis in a 20 μL volume. Thetimetable for the experiment was a follows: on study day 0, intranasalinfection and then on study day 24, early controls were sacrificed forlung burden determination and treatment was started. 28 days postinitiation of treatment (52 days post infection) all treated mice andlate controls were sacrificed for lung burden determination.

For bacterial load determination mice were sacrificed by CO₂asphyxiation. Right lungs were aseptically removed and ground in asealed tissue homogenizer (IdeaWorks! Laboratory Devices, Syracuse,N.Y., USA). The number of viable organisms was determined by serialdilution and titration on 7H10 agar plates. Plates were incubated at 37°C. in ambient air for 4 weeks prior to counting.

TABLE 23a Compound 23A Reduces M. Tuberculosis Burdens in the Mouse M.Tuberculosis 28 Day Lung Infection Model Average Lung Log Reduction LogReduction Burden (Log vs. vs. Treatment Group cfu/lungs) Early ControlLate Control Early Control 4.98 Late Control 5.20 −0.22 (10 mL/KgVehicle) 10 mg/kg BID 5.08 −0.10 0.13 Compound 23A 30 mg/kg BID 4.110.86 1.09 Compound 23A 100 mg/kg BID 3.22 1.76 1.98 Compound 23A 100mg/kg QD 2.94 2.04 2.26 Moxifloxacin

Balb/c mice (6/group) were challenged IN (intranasally) with M.tuberculosis (Erdman; ATCC) at 1×10² cfu/mouse. After 24 days, a singlegroup of mice (Early Control (EC)) was euthanized and the lungsharvested, homogenized and plated to quantitate M. tuberculosis burdens.The additional groups of infected mice were treated via oral gavage withVehicle at 10 ml/kg (10% VitE-TPGS; Late Control, LC) or with Compound23A administered at 10, 30, or 100 mg/kg BID for 28 days. An additionalcontrol group was treated with Moxifloxacin administered at 100 mg/kgQD. After 28 days of treatment, the groups were euthanized and the lungsharvested, homogenized and plated to quantitate M. tuberculosis burdens.Burdens from the right lung for each mouse and the median for each groupof mice were recorded and summarized above in Table 23a.

Results: In summary and as shown above in Table 23a, twice daily oraldosing of Compound 23A exhibited in vivo efficacy against anexperimentally induced lung M. tuberculosis infection in Balb/c mice. 28days of treatment with compound 23A at 30 or 100 mg/kg providedreductions in lung burden vs early controls. In addition, Moxifloxacinprovided lung burden reduction compared to vehicle treated controls.Compound 23A demonstrated dose-dependent reductions of 0.13, 1.09 and1.98 log reductions versus vehicle control (Late) when administered at10, 30, and 100 mg/kg. In addition, doses of 30 and 100 mg/kg ofCompound 23A reduced bacterial burdens versus the early control by0.7-1.5 logs suggesting Compound 23A has bactericidal activity. Thepotent anti-tuberculosis drug Moxifloxacin at 100 mg/kg QD provided lungburden reduction versus the early and late controls as previouslypublished. The reductions were similar to those provided by Compound 23Aadministered at 100 mg/kg indicating that compound 23A has bactericidalactivity against M. tuberculosis.

TABLE 23b Compound W Reduces M. Tuberculosis Burdens in the Mouse M.Tuberculosis 28 Day Lung Infection Model Median Lung Log Reduction LogReduction Burden (Log vs. vs. Treatment Group cfu/lungs) Early ControlLate Control Early Control 4.98 Late Control 4.36 0.62 10 mg/kg BID 4.340.64 0.02 Compound W 30 mg/kg BID 3.00 1.98 1.36 Compound W 100 mg/kgBID 2.35 2.63 2.01 Compound W 100 mg/kg QD 2.94 2.04 1.42 Moxifloxacin

Balb/c mice (6/group) were challenged IN (intranasally) with M.tuberculosis (Erdman; ATCC) at 1×10² cfu/mouse. After 24 days, a singlegroup of mice (Early Control (EC)) was euthanized and the lungsharvested, homogenized and plated to quantitate M. tuberculosis burdens.The additional groups of infected mice were treated via oral gavage withVehicle at 10 ml/kg (10% VitE-TPGS; Late Control, LC) or with Compound Wadministered at 10, 30, or 100 mg/kg BID for 28 days. An additionalcontrol group was treated with Moxifloxacin administered at 100 mg/kgQD. After 28 days of treatment the groups were euthanized and the lungsharvested, homogenized and plated to quantitate M. tuberculosis burdens.Burdens from the right lung for each mouse and the median for each groupof mice were recorded and summarized above in Table 23b.

Results: In summary, and as shown above in Table 23b, Compound Wexhibited robust in vivo efficacy against an experimentally induced M.tuberculosis lung infection in Balb/c mice. After 28 days of treatmentsat 30 and 100 mg/kg BID there were decreases in bacterial densitycompared to early and time-matched vehicle controls. Compound Wdemonstrated dose-dependent reductions of 0.2, 1.36 and 2.02 logreductions versus vehicle control when administered at 10, 30 and 100mg/kg. In addition, doses of 10, 30, and 100 mg/kg BID of Compound Wreduced bacterial burdens versus the early control by 0.64-2.63 logssuggesting Compound W has bactericidal activity against M. tuberculosis.The potent anti-tuberculosis drug Moxifloxacin, at 100 mg/kg QD,provided lung burden reduction versus the early and late controls aspreviously published. The reductions were less than those provided byCompound W administered at 100 mg/kg and similar to those at 30 mg/kgBID Compound W indicating that compound W exhibits anti-tuberculosisactivity on par or better than Moxifloxacin in this assay.

EXAMPLE 39

In Vitro Drug Combination Studies

To evaluate additional potential 2-drug combinations, these are comparedfirst in an in vitro checkerboard experiments performed either incomplete 7H9 broth or in whole blood inoculated with M. tuberculosisH37Rv in log phase growth. Concentrations of 0, 0.25×MIC, MIC, 4×MIC and(if clinically relevant) 16×MIC are tested for each compound.Pyrazinamide combinations may also be examined at pH 6.0, where its MICis 50 μg/ml. For combinations with promising results, a similarcheckerboard may be performed against nutrient-starved M. tuberculosisin PBS, to gain insight into the combination's activity againstnon-replicating organisms. Duplicate wells will be used for eachconcentration pair. Activity will be assessed by quantitative CFU countsperformed after 0 and 7 days of incubation. Samples will be washed withPBS prior to plating.

EXAMPLE 40

In Vitro Drug Combination Studies Using the Whole Blood Assay

The activity of selected 2-drug combinations against intracellularbacilli is also compared in a whole blood culture assay in which bloodfrom healthy volunteers is inoculated with an aliquot of M. tuberculosisand increasing concentrations of drug in a checkerboard fashion similarto that described above. Drug concentrations of 0, 0.25×MIC, MIC, 4×MICand (if clinically relevant) 16×MIC is tested for each drug. Viable CFUcounts are estimated after 0 and 3 days of incubation by washing thecells, osmotically lysing them, inoculating the lysate into MGIT liquidculture bottles and incubating on the cultures on the MGIT system, wherethe time-to-positivity results are applied to a standard curve toestimate the change in log CFU for treatment groups compared topre-treatment and drug-free controls.

EXAMPLE 41

In Vitro Drug Combination Studies Using the Hollow Fiber CartridgeSystem

The hollow fiber cartridge system (HFS) is purchased from FiberCell(Frederick, Md.) and is used for the measurement of bactericidal andsterilizing activity for drug combinations. The peripheral compartmentsof HFS is inoculated with 7.5 log 10 CFU of M. tuberculosis in log-phasegrowth and is incubated at 37° C. under 5% CO2. The peripheralcompartment of each HFS is sampled on days 0, 7, 14, 21, and 28 andsamples are washed twice with normal saline to remove any drugcarryover. The bacterial cultures are inoculated on Middlebrook 7H10agar supplemented with 10% OADC to enumerate the total bacillarypopulation as well as on agar supplemented with either drug orexperimental compound to determine the resistant subpopulations.

Drugs and experimental compounds are administered to the centralcompartment of each HFS via a computer-controlled syringe pump. Forpharmacokinetic matching, drugs and experimental compounds areadministered at the same time to achieve a peak concentration of bothisoniazid and rifampin at 1 h. The central compartments of the HFSs aresampled 12 times during the first 48 h and drug and experimentalcompound concentrations are then measured.

The results of the bactericidal-effect experiments are useful in thedesign of experiments for measuring sterilizing effect. Pharmacokineticand statistical analysis uses the ADAPT 5 program, which has themaximum-likelihood solution via the expectation maximization algorithm.A one-compartment model with first-order input and elimination isutilized and a two-way analysis of variance (ANOVA) with Bonferroniposttest correction is used to compare bacterial burden from triplicateHFSs at each time point in GraphPad Prism version 5.00 (GraphPadSoftware, CA).

EXAMPLE 42

In vivo Mouse M. tuberculosis (Erdman) Lung Infection Model

Animals: female BALB/c mice (5-7 weeks of age; 6/group), are obtainedfrom Jackson Laboratories, (Bar Harbor, Me.) and are housed andmaintained in a BSL3 facility in accordance with the Guide to the Careand Use of Experimental Animals.

Bacterial strain and stocks

M. tuberculosis ATCC 35801 (strain Erdman) is obtained from the ATCC(Manassas, Va., USA). The organism is grown in 20 tubes of modified 7H10broth (pH 6.6; 7H10 agar formulation with agar and malachite greenomitted) with 10% OADC (oleic acid, albumin, dextrose, catalase)enrichment (BBL Microbiology Systems, Cockeysville, Md., USA) and 0.05%Tween 80 for 5-10 days on a rotary shaker at 37′C. The cultures arepooled and diluted to 100 Klett units [equivalent to 5×10⁷ colonyforming units (cfu)/mL] (Photoelectric Colorimeter; Manostat Corp., NewYork, N.Y., USA). The culture is aliquotted and frozen at −70′C. On theday of infection, the culture is thawed and the final inoculum isdetermined. The final inoculum size is determined by diluting to 5×10⁻²and plating 0.1 mL, in triplicate, on 7H10 agar plates (BBL MicrobiologySystems) supplemented with 10% OADC enrichment. The plates are incubatedat 37′C in ambient air for 4 weeks.

Mouse M. tuberculosis (Erdman) Infection Model

Intranasal infection groups of mice are anaesthetized by intramusculardelivery of a telazol (45 mg/kg)/xylazine (7.5 mg/kg) cocktail (LederleParenterals, Carolina, Puerto Rico and Bayer Corp., Shawnee Mission,Kans., USA, respectively) and subsequently infected intranasally with˜10² viable M. tuberculosis in a 20 μL volume. The timetable for theexperiment is as follows: day 0, intranasal infection; on study day 24early controls are sacrificed for lung burden determination andtreatment is started. 28 days post initiation of treatment 52 days postinfection, all treated mice and late controls are sacrificed for lungburden determination.

For bacterial load determination mice are sacrificed by CO2asphyxiation. Right lungs are aseptically removed and ground in a sealedtissue homogenizer (IdeaWorks! Laboratory Devices, Syracuse, N.Y., USA).The number of viable organisms is determined by serial dilution andtitration on 7H10 agar plates. Plates are incubated at 37° C. in ambientair for 4 weeks prior to counting.

Groups for three compound combination studies:

Early Controls

Late Controls

TMC-207 25 mg/kg+pyrazinamide 150 mg/kg+Rifapentine 10 mg/kg

TMC-207 25 mg/kg+Compound (IB) where X is PO(OH)₂ 100 mg/kg+pyrazinamide150 mg/kg

TMC-207 25 mg/kg+Moxifloxacin 100 mg/kg+pyrazinamide 150 mg/kg

Compound (IB) where X is PO(OH)₂ 100 mg/kg+Rifapentine 10mg/kg+pyrazinamide 150 mg/kg

Moxifloxacin 100 mg/kg+Rifapentine 10 mg/kg+pyrazinamide 150 mg/kg

Compound (IB) where X is PO(OH)₂100 mg/kg+linezolid 100mg/kg+pyrazinamide 150 mg/kg

Compound (IB) where X is PO(OH)₂100 mg/kg+clofazimine 20mg/kg+pyrazinamide 150 mg/kg

Compound (IB) where X is PO(OH)₂ 100 mg/kg+Moxifloxacin 100mg/kg+pyrazinamide 150 mg/kg

Formulation Preparation:

Group #3: TMC-207 is formulated in 20% Hydroxypropyl-B-Cyclodextrin andtreatment occurs in the morning. In the afternoon, (minimum of 2 hrsbetween dosing) all other compounds are combined for the groups of micein one tube and dissolve by first adding 50% polyethylene glycol untildissolved and then adding 50% ddH₂O.

BALB/c mice (6/group) are challenged IN with M. tuberculosis (Erdman;ATCC) at a dose of 1×10² cfu/mouse. After 24 days a single group of mice(Early Control (EC)) is euthanized and the lungs harvested, homogenizedand plated to quantify M. tuberculosis burdens. Compounds areadministered at 10, 30, or 100 mg/kg BID for 28 days. After 28 days oftreatment the groups are euthanized and the lungs harvested, homogenizedand plated to quantify M. tuberculosis burdens.

REFERENCES

-   -   Combinations of antibiotics and nonantibiotic drugs enhance        antimicrobial efficacy. Ejim L, Farha M A, Falconer S B,        Wildenhain J, Coombes B K, Tyers M, Brown E D, Wright G D. Nat        Chem. Biol. 2011 June; 7(6):348-50.    -   Selection of a moxifloxacin dose that suppresses drug resistance        in Mycobacterium tuberculosis, by use of an in vitro        pharmacodynamic infection model and mathematical modeling. Gumbo        T, Louie A, Deziel M R, Parsons L M, Salfinger M, Drusano G L. J        Infect Dis. 2004 Nov. 1; 190(9):1642-51.    -   Pharmacokinetics and whole-blood bactericidal activity against        Mycobacterium tuberculosis of single doses of PNU-100480 in        healthy volunteers. Wallis R S, Jakubiec W M, Kumar V, Silvia A        M, Paige D, Dimitrova D, Li X, Ladutko L, Campbell S, Friedland        G, Mitton-Fry M, Miller P F. J Infect Dis. 2010 Sep. 1;        202(5):745-51.

EXAMPLE 43

Evaluation of the Anti-Tuberculosis Activity of Compounds in Mice

PHASE 1—Evaluation of Compounds as Monotherapy Against Established TB inMice

Methods

The experimental scheme is presented in Table 24. BALB/c mice will beinfected with ˜100 CFU of virulent M. tuberculosis H37Rv in order toproduce a stable infection with M. tuberculosis of ˜10⁶ organisms in thelung at the initiation of treatment 5 weeks later (D0). Drugs will beprepared in an appropriate vehicle. Treatment will be administereddaily, 5 days per week, by esophageal gavage unless subcutaneousinjection is required. Outcomes will be lung CFU counts after 4 weeks oftreatment. Quantitative cultures of lung samples will be performed induplicate on OADC-enriched 7H11 agar medium. Group mean differences inlung CFU counts will be compared using one-way ANOVA with Dunnett'spost-test (GraphPad Prism 4) to adjust for multiple comparisons.

Explanation of Treatment Groups

Untreated: This is the negative control group. Five mice will besacrificed the day after M. tuberculosis infection (D-34) and on the dayof treatment initiation (D0) to determine the number of bacilliimplanted and the extent of multiplication from D-35 to D0,respectively. Additional mice will be sacrificed 4 weeks formicrobiological characterization of the natural history of infection.

Isoniazid (INH): mice in this control group will receive this first-linedrug known for its strong bactericidal activity against activelymultiplying organisms but reduced activity against non-activelymultiplying organisms.

Rifampin (RIF): mice in this control group will receive this first-linedrug known for its strong bactericidal activity against non-activelymultiplying organisms.

Test compound A (A): mice in this group will receive a first compound offormula (I) (“A”) at one of 3 doses.

Test compound B (B): mice in this group will receive a second compoundof formula (I) (“B”) at one of 3 doses.

TABLE 24 Experimental scheme for dose-ranging activity study No of micekilled by time point Regimen [dose(mg/kg)] D-35 D0 Wk 4 Total Untreated5 5 5 15 INH (10) 5 5 RIF (10) 5 5 A (10) 5 5 A (30) 5 5 A (100) 5 5 B(10) 5 5 B (30) 5 5 B (100) 5 5 Total 5 5 45 55

The experiment will also include a PK study to determine the 24-hourserum and lung PK profile for each test compound and dose in infectedmice during the 2nd week of treatment. Mice will be sacrificed accordingto the scheme in Table 25, around the dose administered on Wednesday orThursday during the 2nd week of treatment. Three mice will be sacrificedfor each drug and dose at the indicated time points before and afterdrug administration. At the time of sacrifice, mice will be anesthetizedwith isoflurane, using the drop method, and exsanguinated by cardiacpuncture. Serum will be harvested and frozen at −80° C. The right lungwill be harvested, homogenized thoroughly and frozen at −80° C.

TABLE 25 Scheme for serum and lung PK sub-study No of mice killed bytime point 0 h 0.5 h 1 h 2 h 4 h 8 h Total 3 3 3 3 3 3 18

To perform serum and lung PK for all 3 doses of both drugs, a total of108 mice will be required.

PHASE 2—Evaluation of Compound Activity in Combination with Existing TBDrugs

1) Experiment to identify the best companion drugs for the testcompounds

The interaction of Compounds A and/or B with existing TB drugs will beevaluated first in 2 in vitro models to inform the design of long-termcombination therapy studies with 3- and/or 4-drug combinations thatutilize relapse rate as the measure for stable cure and thereby promotethe most efficient use of limited resources.

Methods

In Vitro Checkerboard Assay

Potential 2-drug combinations will be compared first in in vitrocheckerboard experiments performed either in complete 7H9 broth or inwhole blood inoculated with M. tuberculosis H37Rv in log phase growth.Drug concentrations of 0, 0.25×MIC, MIC, 4×MIC and (if clinicallyrelevant) 16×MIC will be tested for each drug. PZA will be evaluated atnormal pH, where its MIC against M. tuberculosis H37Rv is 250 μg/ml. Itmay also be examined at pH 6.0, where its MIC is 50 μg/ml. Forcombinations with promising results, a similar checkerboard may beperformed against nutrient-starved M. tuberculosis in PBS, to gaininsight into the combination's activity against non-replicatingorganisms.

A sample experimental scheme for a checkerboard experiment is presentedin Table 26. Duplicate wells will be used for each concentration pair.Activity will be assessed by quantitative CFU counts performed after 0and 7 days of incubation. Samples will be washed with PBS prior toplating.

In Vitro Whole Blood Assay

Activity of selected 2-drug combinations against intracellular bacilliwill be compared in a whole blood culture assay in which blood fromhealthy volunteers is inoculated with an aliquot of M. tuberculosis andincreasing concentrations of drug in a checkerboard fashion similar tothat described above. Drug concentrations of 0, 0.25×MIC, MIC, 4×MIC and(if clinically relevant) 16×MIC will be tested for each drug, asdepicted in Table 26. Viable CFU counts are estimated after 0 and 3 daysof incubation by washing the cells, osmotically lysing them, inoculatingthe lysate into MGIT liquid culture bottles and incubating on thecultures on the MGIT system, where the time-to-positivity results areapplied to a standard curve to estimate the change in log CFU fortreatment groups compared to pre-treatment and drug-free controls.

Rationale for Drugs to be Tested

Isoniazid (INH): first-line drug known for its strong bactericidalactivity against actively multiplying organisms but reduced activityagainst non-actively multiplying organisms.

Rifampin (RIF): first-line drug known for its moderate activity againstactively multiplying organisms, but strong bactericidal activity againstnon-actively multiplying organisms (sterilizing activity).

Pyrazinamide (PZA): first-line drug known for having pH-dependentactivity against M. tuberculosis in vitro, but significant sterilizingactivity in mice, presumably against M. tuberculosis inside activatedmacrophages.

Moxifloxacin (MXF): key second-line drug known for its strongbactericidal activity against actively multiplying organisms but reducedactivity against non-actively multiplying organisms. MXF is beingstudied in clinical trials to evaluate whether it belongs as afirst-line TB drug.

Linezolid (LZD): second-line drug commonly used in salvage therapy forrecalcitrant drug-resistant TB. LZD may also serve as a surrogate fornew oxazolidinones in clinical development.

Cmpd A (A): Test compound A.

Cmpd B (B): Test compound B.

TABLE 26 Sample scheme for checkerboard experiment Conc of Concentrationof Drug 1 Drug 2 0 0.25x MIC MIC 4x MIC 16x MIC 0 0.25x MIC MIC 4x MIC16x MIC

2) Experiment to evaluate the sterilizing activity of novel combinationsincluding test compounds of formula (I).

At present, the gold standard for measuring a regimen's sterilizingactivity in the mouse model is the assessment of relapse afterdiscontinuation of therapy. With available regimens as standardcomparators, such experiments require 7-10 months to complete due to therequirement for a treatment-free follow-up period of ≧3 months todetermine the proportion of mice with culture-positive relapse. Becauseof the time- and cost-consuming nature of such experiments, it isimperative to utilize the most efficient study designs possible. Byestablishing the relative activity of various 2-drug building blocks inthe short-term infection model as described above, the most promisingregimens can be carried forward into 1 or 2 relapse-based studies tocompare the activity of such regimens to that of the standard first-lineregimen and/or that of more potent experimental regimens.

A sample experimental scheme is presented in Table 27. In this example,the effect of adding test compound A to the first-line regimen orsubstituting it for INH is examined, as is the substitution of testcompound B for ethambutol or amikacin in an idealized second-lineregimen comprised of existing drugs. In each case, the regimens inquestion are truncated after treatment of reduced duration in order todemonstrate whether the incorporation of test compound could have atreatment-shortening effect. The primary endpoint is the proportion ofmice with positive cultures (i.e., relapse) 3 months after thediscontinuation of therapy. Mice will be infected by the aerosol routewith approximately 4 log₁₀ CFU on Day −14. The infection will incubatefor 14 days before mice are randomized into treatment groups asindicated and treatment is begun. The indicated regimens will beadministered as described above. Group mean lung CFU counts will becompared by one-way ANOVA with Bonferroni's post-test to adjust formultiple comparisons. Additional cohorts of 15 mice will be held for 3months after completing various durations of treatment before beingsacrificed for relapse determination. The entire lung homogenate will beplated on 7H11 agar. The proportions of mice with culture-positiverelapse will be compared using Fisher's Exact test with adjustment formultiple corrections.

TABLE 27 Example of an experimental scheme to assess the sterilizingactivity of promising regimens containing test compounds Time point andNo. of mice to sacrifice** Regimen* D-13 D0 M2 M4 (+3) M5 (+3) M6 (+3)Total 2RHZ/3RH 6 6 5 5(15) 5(15) 57 2RHZA/3RHA 5 5(15) 5(15) 45 2RAZ/3RA5 5(15) 5(15) 45 2MEZAmk/4ME 5 5 5(15) 5(15) 50 2MBZAmk/4MB 5 5 5(15)5(15) 50 2MEZB/4MEB 5 5 5(15) 5(15) 50 Total 6 6 30  30(45) 30(90) 15(45)  297 *R, RIF 10 mg/kg; H, INH 10 mg/kg; Z, PZA 150 mg/kg; A, Testcompound A; B, Test compound B; M, MXF 100 mg/kg; E, ethambutol 100mg/kg; Amk, amikacin 30 mg/kg **Time points are shown in days (D)(day-13 [D-13] pr day 0 [D0]) or monthls (M) (eg., 2 months = M2) oftreatment. (+3) indicates that the mice are held for 3 months after thecompletion of treatment at the indicated time point. Explanation oftreatment groups 2RHZ/3RH: first-line regimen control consisting of 2months of RIF, INH, and PZA, followed by 3 months of RIF and INH.2RHZA/3RHA: test regimen in which test compound A is added to thefirst-line regimen. 2RAZE/3RA: test regimen in which test compound A issubstituted for INH in the first-line regimen. 2MEZAmk/4ME: second-lineregimen control consisting of 2 months of MXF, ethambutol, PZA andamikacin, followed by 4 months of MXF and ethambutol. 2MEZAmk/4ME: testregimen in which test compound B is substituted for ethambutol in thesecond-line regimen. 2MEZAmk/4ME: test regimen in which test compound Bis substituted for amikacin in the second-line regimen.

EXAMPLE 44

Minimal Inhibitory Concentration (MIC) of Compound 23A in M.tuberculosis (“Mtb”) Bacterial Broth Culture Against Diverse MtbIsolates and Various Species of Mycobacteria

To determine the MIC of Compound 23A, 96-well microtiter plates wereused (Corning #3904) to culture M. tuberculosis in Middlebrook 7H9 broth(BD271310) containing ADC enrichment, while agar plates were used tosub-culture various isolates of Mtb streaked to single colonies. Cellsuspensions were made containing ˜10⁸ cells/ml following sonication andthen diluted 1/200 by transferring 0.2 ml of cells to 40 ml sterile 7H9broth with ADC supplement (a final concentration of ˜10⁶ cells/ml). 100μl of Mtb cells were then (˜5×10⁴ cells) added each microtiter wellcontaining 1 μl of test compounds in DMSO (see below). Microtiter plateswere incubated in a humidified 37° C. chamber for 9 days and bacterialgrowth was measured by either visual inspection or by adding 30 μl of0.01% sterile resazurin to each well and measuring the backgroundfluorescence at an Excitation of 492 nm/emission of 595 nm after 24hours. The minimum inhibitory concentrations (MICs) were defined as thelowest concentration of an antimicrobial that inhibits the growth ofbacteria by ≧70%.

TABLE 28 MICs of Compound 23A and Moxifloxacin (as a comparator) in anexpanded set of Mycobacterium tuberculosis (Mtb) strains Organism 23AMoxifloxacin Mtb H37Rv 0.015, 0.015 0.03, 0.03 Mtb Erdman 0.06, 0.060.03, 0.03 Mtb CDC1551 0.015, 0.015 0.015, 0.015 Mtb HN878 (Beijing-0.015, 0.015  0.03, 0.015 Type) Mtb GN9 0.03, 0.03  0.03, 0.015 M. avium103 0.12, 0.23 NT M. avium Far 0.23, 0.23 NT M. avium 3404.4 0.23, 0.23NT M. kansasii 303 0.03, 0.03 0.03, 0.03 M. kansasii 316 0.06, 0.060.03, 0.03 M. kansasii 379 <0.015, <0.015 0.03, 0.03 M. smegmatis 6.25NT Conclusion: Compound 23A is potently active against a diverse rangeof Mtb species.

TABLE 29 MICs (ug/ml) of Compound 23A in Drug Resistant Mycobacteria inBroth Culture (again using Moxifloxacin as comparator) Organism Cmpd 23AMoxifloxacin M. tuberculosis Erdman 0.125, 0.06   0.03, 0.015 M.tuberculosis Levo^(R) 2D 0.015, 0.015 2, 2 M. tuberculosis Levo/Gat^(R)0.06, 0.06 8, 8 2C M. tuberculosis 5 0.125, 0.125 8, 4 M. tuberculosisLevo^(R) 2D and M. tuberculosis Levo/Gat^(R) 2C are laboratory derivedresistant strains of M. tuberculosis Erdman; they are single-drugresistant strains. M. tuberculosis 5 is an XDR strain Conclusion:Compound 23A is also active against drug resistant Mtb

EXAMPLE 45

MIC Determination in the Low-Oxygen-Recovery Assay (LORA) forNonreplicating M. tuberculosis

Screening for new antimicrobial agents is routinely conducted onlyagainst actively replicating bacteria. However, it is now widelyaccepted that a physiological state of non-replicating persistence (NRP)is responsible for antimicrobial tolerance in many bacterial infections.In tuberculosis, the key to shortening the 6-month regimen lies intargeting this NRP subpopulation. Therefore, Compound 23A was tested ina high-throughput, luminescence-based low oxygen-recovery assay (LORA),developed to screen antimicrobial agents against NRP Mycobacteriumtuberculosis as described by Cho et al 2007. Cho S H, Warit S, Wan B,Hwang C H, Pauli G F, Franzblau S G; “Low-Oxygen-Recovery Assay forHigh-Throughput Screening of Compounds against NonreplicatingMycobacterium tuberculosis” Antimicrob Agents Chemother. 2007 April;51(4): 1380-1385 doi: 10.1128/AAC.00055-06.

Results:

The LORA MIC for Compound 23A was 0.25 ug/mL, indicating that the testcompound retained activity against non-replicating, persistentMycobacterium tuberculosis. In contrast Moxiflxacin was inactive (MIC>40ug/ml) and gatifloxacin was 10-fold less active (MIC=3.6 ug/ml)

EXAMPLE 46

Efficacy Against Intracellular M. tuberculosis

Cultures of recombinant M. tuberculosis (Mtb) expressing luciferase weremaintained in 7H9 broth supplemented with 0.05% Tween 80, 10% ADC, and20 ug/ml kanamycin, (25 ml in a filter-cap 125 ml plastic Erlenmeyerflask), at 37° C. with static incubation. Immediately prior to infectionof macrophages or cell lines, the Mtb cultures were sonicated insuspension for 10 seconds and diluted to a density of 8×10⁵ cells/ml fora multiplicity of infection (MOI) of 2:1. Prior to M. tuberculosisinfection, PMA-activated THP-1 or J774 cells were adjusted to a celldensity of 2−3×10⁵ cells/ml in 25 mM HEPES-buffered RPMI-1640 media(without phenol red; medium #2) supplemented with 10% FBS, L-glutamine,and 0.05 mM b-mercaptoethanol.

Test compounds were dispensed into sterile round bottom 96 well tissueculture plates at the desired concentration in 1 μl volumes of DMSO(0.5% DMSO final). Just prior to treatment of Mtb-infected cells withthe compound, supernatant containing un-ingested Mtb from each well wasremoved and replaced with 100 μl fresh media. Cell cultures wereincubated remaining plates at 37° C., 5% CO₂, in humidified chamber andthe endpoint luciferase of all test and control plates was determined 5days (120 hours) after infection by adding 100 ul of Bright Glow reagentto each well, incubating for 10 minutes at room temperature, coveringwith adhesive top seal and reading the luminescence in TecanSpectrafluor+, at a gain of 150 at maximum integration time. IC₅₀ wasdefined the concentration of an antimicrobial that inhibited the growthof intracellular bacteria by 50%

Results: The effects of Compound 23A on intracellular replication on Mtbwere examined in two different cell types (THP-1 and J774) and threedifferent Mtb strains (Erdman, H37Ra and CDC1551). Three othergyrase-target antibiotics were used as controls. Compound 23A wasefficacious against all three intracellular Mtb strains in both celltypes.

TABLE 30 Mtb Strain/Cell type 23A Moxifloxacin Gatifloxacin NovobiocinErdman (MIC) 0.06 0.03 0.25 61.00 Mtb H37Ra in 0.03 0.33 0.20 2.10 THP1Cells (IC₅₀) Mtb CDC1551 in 0.12 0.21 0.94 NT J774 Cells (IC₅₀) MtbH37Rv in 0.21 NT NT NT J774 Cells (IC₅₀) Mtb Erdman in <0.17 NT NT NTJ774 Cells (IC₅₀)

TABLE 31 Comparison of Compound 23A vs. Standard TB Drugs in Culture andIntracellular Macrophage (THP1) Assay MIC (μg/mL) Compound BrothMacrophage Cells 23A 0.01 0.03 Isoniazid 0.21 0.012 Rifampin 0.03 0.008Ethambutol 2.55 1.26 Pyrazinamide >12.3 >2.5 Linezolid 0.53 0.37Moxifloxacin 0.12 0.33 Mtb H37Ra was used as the test strain. The datashow that Compound 23A has at least equivalent potency as approvedanti-TB agents.

EXAMPLE 47

Efficacy of Compound 23A in Combination with Approved TB Agents

A checkerboard approach was used to determine the effect ofco-administration of Compound 23A with approved anti-TB agents, usingboth broth culture and Intracellular. In order to assess whether theeffects were antagonistic, additive or synergistic the FractionalInhibitory Concentration (FIC) Index was calculated as follows:

${FIC}_{index} = {\frac{{MIC}_{A}\mspace{14mu}{with}\mspace{14mu} B}{{MIC}_{A}} + \frac{{MIC}_{B}\mspace{14mu}{with}\mspace{14mu} A}{{MIC}_{B}}}$

The combination is considered antagonistic if the FIC_(index) is greatthen 4, indifferent or additive if the FIC_(index) between 0.5 and 4,and synergistic if the index is less index is an than 0.5. The resultsare tabulated below and show that Compound 23A was generally additive tothe effect of approved anti-TB agents and may be synergistic when usedin combination with TMC207 and Clofazimine. None of the drugcombinations used showed antagonistic effects.

TABLE 32 Cmpd 23A Mtb in Broth Culture Intracellular Mtb with AdditiveSynergy Additive Synergy Isoniazid 0.75 0.75 Rifampin 0.63 2.00Moxifloxacin 1.00 1.00 TMC-207 1.00 0.50 Amikaicin 2.00 2.00 Linezolid*0.50 2.00 PA-824 2.00 1.00 Ethambutol 0.63 2.00 Clofazimine 2.00 0.50

What is claimed is:
 1. A method of controlling, treating or reducing theadvancement, severity or effects of a mycobacterium disease comprisingadministering to a patient in need thereof a therapeutically effectiveamount of: (a) a compound of formula

wherein R is hydrogen or fluorine; X is hydrogen, —PO(OH)2, —PO(OH)O⁻M⁺,—PO(O⁻)₂.2M⁺, or —PO(O⁻)₂.D²⁺; M⁺ is a pharmaceutically acceptablemonovalent cation; and D²⁺ is a pharmaceutically acceptable divalentcation; or a pharmaceutically acceptable salt thereof; in combinationwith: (b) one or more antibiotic compounds comprising a diarylquinolone,rifapentine, rifalazil, a nitroimidazole, a benzothiazinone,capreomycin, clofazimine, cycloserine, dapsone, a thiocarbamide,ethambutol, DC-159a, a nitrobenzthiazole, sutezolid (PNU-100480),AZD-5847, posizolid (AZD-2563), para-aminosalicylic acid, SQ-109,SQ-609, a capuramycin, a caprazene nucleoside, an isothiazoloquinolone,thioridazine, thiacetazone, dirithromycin, roxithromycin, telithromycin,azithromycin, clarithromycin, erythromycin, amikacin, kanamycin,streptomycin, levofloxacin, moxifloxacin, gatifloxacin, linezolid,rifalazil, meropenem, clavulanate, PA 824, pyrazinamide, TMC-207,oxazolidinine, nitroimidazole, or isoniazid, with the proviso that whenthe one or more antibiotic compounds is thioridazine, azithromycin,clarithromycin, erythromycin, amikacin, kanamycin, streptomycin,levofloxacin, moxifloxacin, gatifloxacin, linezolid, rifalazil,meropenem, clavulanate, or isoniazid, then an additional antibiotic isalso present in the combination.
 2. The method of claim 1, wherein saidone or more antibiotic compounds comprises bedaquiline, rifapentine,moxifloxacin, linezolid, delaminid, or PA
 824. 3. The method of claim 1,wherein said one or more antibiotic compounds comprises moxifloxacin,linezolid, rifalazil, meropenem, clavulanate, pyrazinamide, andisoniazid.
 4. The method of claim 1 wherein at least one of theantibiotics is pyrazinamide.
 5. The method of claim 1 wherein thecompound of formula (I) is a compound of formula

wherein X is —PO(OH)₂, —PO(OH)O⁻M⁺, —PO(O⁻)₂.2M⁺, or —PO(O⁻)₂.D²⁺; M⁺ isa pharmaceutically acceptable monovalent cation; and D²⁺ is apharmaceutically acceptable divalent cation; or a pharmaceuticallyacceptable salt thereof.
 6. The method of claim 1 wherein the compoundof formula (I) is a compound of formula

wherein R is hydrogen or fluorine; or a pharmaceutically acceptable saltthereof.
 7. The method of claim 1 wherein the compound of formula (I) is(R)-1-ethyl-3-(5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea, or a pharmaceutically acceptable saltthereof.
 8. The method of claim 1, wherein the compound of formula (I)is(R)-1-ethyl-3-(6-fluoro-5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea,or a pharmaceutically acceptable salt thereof.
 9. The method of claim 1,wherein the salt of formula (I) is a methanesulfonic acid salt of(R)-1-ethyl-3-(5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea.10. The method of claim 1, wherein the salt of formula (I) is amethanesulfonic acid salt of(R)-1-ethyl-3-(6-fluoro-5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea.11. The method according to claim 1, wherein the compound of formula (I)is disodium(R)-2-(5-(2-(3-ethylureido)-6-fluoro-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)pyrimidin-2-yl)propan-2-yl phosphate.
 12. The methodaccording to claim 1 wherein the mycobacterial disease is caused by M.tuberculosis, M. avium intracellulare, M. ulcerans, M. kansasii, M.fortuitum, M. abcesses, M. leprae, M. africanum, M. marinum, M. aviumparatuberculosis, or M. bovis, M. chelone, M. scrofulaceum, M. xenopi,M. intracellulare, or M. microti.
 13. A method according claim 1 whereinthe compound of formula (I) is administered with only one antibioticselected from rifapentine, TMC-207, SQ-109, a nitroimidazole, or anoxazolidinone.
 14. A method according to claim 13 wherein the antibioticis rifapentine.
 15. A method according claim 13 further comprisingadministering pyrazinamide.
 16. A method according to claim 13 whereinthe compound of formula (I) is(R)-1-ethyl-3-(6-fluoro-5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea,or a pharmaceutically acceptable salt thereof.
 17. A method of treatingtuberculosis comprising administering a therapeutically effective amountof: (a) a compound of formula (I):

wherein R is hydrogen or fluorine; X is hydrogen, —PO(OH)₂, —PO(OH)O⁻M⁺,—PO(O⁻)₂.2M⁺, or —PO(O⁻)₂.D²⁺; M⁺ is a pharmaceutically acceptablemonovalent cation; and D²⁺ is a pharmaceutically acceptable divalentcation; or a pharmaceutically acceptable salt thereof; in combinationwith: (b) one or more antibiotic compounds comprising a diarylquinolone,rifapentine, rifalazil, a nitroimidazole, a benzothiazinone,capreomycin, clofazimine, cycloserine, dapsone, a thiocarbamide,ethambutol, DC-159a, a nitrobenzthiazole, sutezolid (PNU-100480),AZD-5847, posizolid (AZD-2563), para-aminosalicylic acid, SQ-109,SQ-609, a capuramycin, a caprazene nucleoside, an isothiazoloquinolone,thioridazine, moxifloxacin, gatifloxacin, linezolid, rifalazil,meropenem, clavulanate, or isoniazid, with the proviso that when the oneor more antibiotic compounds is thioridazine, moxifloxacin,gatifloxacin, linezolid, rifalazil, meropenem, clavulanate, ad orisoniazid, then an additional antibiotic is also present in thecombination.
 18. The method of claim 1 wherein the one or moreantibiotic compounds are TMC-207 and pyrazinamide.
 19. The method ofclaim 1 wherein the one or more antibiotic compounds are rifapentine andpyrazinamide.
 20. The method of claim 1 wherein the one or moreantibiotic compounds are oxazolidinone and pyrazinamide.
 21. The methodof claim 1 wherein the one or more antibiotic compounds arenitroimidazole and pyrazinamide.
 22. The method of claim 1 wherein theone or more antibiotic compounds are pyrazinamide and SQ-109.
 23. Themethod of claim 1 wherein the one or more antibiotic compounds areTMC-207, SQ-109, and pyrazinamide.
 24. The method of claim 1 wherein theone or more antibiotic compounds are SQ-109, clavulanate and meropenem.25. The method of claim 1 wherein the one or more antibiotic compoundsare rifapentine, SQ-109, and pyrazinamide.
 26. The method of claim 1wherein the one or more antibiotic compounds are oxazolidinone, SQ-109,and pyrazinamide.
 27. The method of claim 1 wherein the one or moreantibiotic compounds are nitroimidazole, SQ-109, and pyrazinamide. 28.The method of claim 1 wherein the one or more antibiotic compounds areclavulanate, meropenem, and SQ-109.